NADPH oxidase mutant and its application

Abstract

The present invention discloses a NADPH oxidase mutant and its application, belonging to the field of bioenzyme engineering technology. The mutant is obtained by screening through homology modeling technology and site-directed saturation mutagenesis technology on the basis of SmNOX; the mutant is obtained by site-directed mutagenesis of the NADPH oxidase with an amino acid sequence such as that shown in SEQ ID NO:2, and the point mutation is T114E, I116F or P118S, and the corresponding amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8. The unit enzyme activity of the three SmNOX enzyme mutants obtained by the present invention is 4.6 to 6.0 U / mg, which is 1.4 to 1.9 times higher than that of the original SmNOX enzyme, and can be more effectively used in related redox reactions, and has good application prospects in the fields of pharmaceuticals, food, fine chemicals, pesticides, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of bioenzyme engineering and relates to a NADPH oxidase, in particular to a NADPH oxidase mutant and an application thereof. Background Art

[0002] Oxidoreductase is an important class of biocatalysts, which has important uses in the fields of pharmaceuticals, food, fine chemicals, pesticides, etc. It is mainly used to prepare chiral compounds, such as hydroxy acids, amino acids, steroids, chiral alcohols, etc. When oxidoreductase is performing biocatalysis, coenzymes (NAD, NADP) play the role of electron transfer. As the reaction continues, the coenzymes will gradually decrease, eventually leading to the termination of the reaction. To continue the reaction, it is necessary to constantly replenish the coenzymes, so the efficient supply of coenzymes is one of the key technologies for the development of oxidoreductase catalytic reactions. However, coenzymes are expensive, and it is obviously impractical to add a large amount of supplementary coenzymes during the production process. Therefore, from the perspective of the economic benefits of industrial applications, in addition to having suitable enzymes and reaction engineering technologies, oxidoreductases must also provide an efficient and low-cost coenzyme regeneration system in their applications.

[0003] Coenzyme regeneration is to regenerate the coenzyme from the oxidized state to the reduced state, or vice versa, so as to keep the coenzyme at a certain catalytic level. In order to solve the problem of coenzyme regeneration, a series of methods have been proposed, including chemical, photochemical, enzymatic and electrochemical regeneration systems. Among them, the enzymatic regeneration system has the advantages of mild reaction conditions, low catalytic efficiency and environmental friendliness, and has received widespread attention. Among the common oxidized coenzyme regeneration systems, NAD(P)H oxidase is one of the most promising oxidative coenzyme regeneration systems, because NAD(P)H oxidase can directly use molecular oxygen as an oxidant, without the need for other auxiliary substrates or mediators, thereby reducing costs; the product is water or hydrogen peroxide, and there is no need to deal with by-products or the by-products are simple to deal with, which is very conducive to application in industrial production.

[0004] The present invention aims to develop an oxidase for efficient regeneration of NADP+, and selects NAD(P)H-specific Streptococcus mutans NAD(P)H oxidase 2 (SmNOX) as a template for mutation. The reason for selecting SmNOX is that it is stable, highly active, does not require additional addition of FADH or DTT, and can be efficiently expressed in Escherichia coli. At the same time, SmNOX can directly use oxygen to directly regenerate the coenzyme NADP, and is one of the few NADH oxidases that produces water instead of hydrogen peroxide, and has no side reaction products, so it is very suitable for use in constructing a coenzyme circulation system.

[0005] Point saturation mutagenesis is an emerging technology in protein engineering. It is a technique that modifies the coding gene of the target protein to obtain mutants in which the amino acids at the target site are replaced by 19 other natural amino acids in a short period of time. It is currently widely used in protein modification and structure-function relationship research. For example, point saturation mutagenesis is used to identify protein functional sites, improve enzyme specific activity, and improve enzyme thermal stability, substrate binding specificity, stereoisomer specificity and other properties. The present invention uses point saturation mutagenesis combined with homology modeling technology to modify SmNOX, thereby obtaining mutants that efficiently and stably regenerate NADP+. Summary of the invention

[0006] The object of the present invention is to provide a NADPH oxidase mutant with improved catalytic activity and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a NADPH oxidase mutant, wherein the NADPH oxidase mutant is obtained by site-directed mutagenesis of the NADPH oxidase in the amino acid sequence as shown in SEQ ID NO:2, the point mutation is T114E, I116F or P118S, and the corresponding amino acid sequence of the NADPH oxidase mutant is shown in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8.

[0008] The NADPH oxidase mutant of the present invention is obtained by mutation screening of NADH oxidase 2 (SmNOX) from Streptococcus mutans, wherein the nucleotide sequence of the NADH oxidase 2 (SmNOX) is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The gene sequence of SmNOX is synthesized by Changzhou Jiyu Biotechnology Co., Ltd., and the gene sequence is codon-optimized according to the codon preference of Escherichia coli.

[0009] Through site-directed saturation mutagenesis technology, the target amino acid was replaced by 19 other amino acids, and mutants with significantly improved enzyme activity were screened out and the mutation sites were verified by sequencing. The homology modeling of Streptococcus mutans NADH oxidase 2 was based on the X-ray structure of NADH oxidase from Streptococcus pyogenes, and the consistency between the sequences was 77.5%. The sites related to NADPH binding were predicted to be 114, 116 and 118, and after site-directed saturation mutagenesis of these three sites, mutants with improved substrate conversion rate were screened. The screening experiment results showed that when the isoleucine (I) at site 116 was mutated to phenylalanine (F), or when the proline (P) at site 118 was mutated to serine (S), or when the threonine (T) at site 114 was mutated to glutamic acid (E), the catalytic activity of the mutant was significantly improved.

[0010] The present invention also claims to protect the coding gene of the NADPH oxidase mutant.

[0011] Based on the amino acid sequence of the NADPH oxidase mutant provided in the present invention, it is not difficult for a person skilled in the art to obtain the corresponding coding gene sequence according to the coding rules of codons.

[0012] As a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is shown as SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

[0013] The present invention further provides the nucleotide sequence of the above-mentioned coding gene, which can be effectively expressed in general engineering bacteria, including Escherichia coli, Pichia pastoris, Streptomyces, Bacillus subtilis, etc.

[0014] The present invention also claims to protect the recombinant expression vector containing the encoding gene.

[0015] Based on the nucleotide sequence of the NADPH oxidase mutant provided in the present invention, it is not difficult for a person skilled in the art to access the encoding gene into a corresponding expression vector through existing genetic engineering methods.

[0016] As a preferred embodiment of the present invention, the vector backbone of the recombinant expression vector includes one of pET, pCW, pUC and pPIC9k.

[0017] The present invention also claims to protect the recombinant cells containing the encoding gene.

[0018] The expression vector can be transformed into a suitable host cell to obtain the corresponding recombinant cell for expressing the target protein.

[0019] As a preferred embodiment of the present invention, the host cell used by the recombinant cell includes one of Escherichia coli, Pichia pastoris, Streptomyces and Bacillus subtilis.

[0020] Furthermore, the present invention also claims to protect the use of the NADPH oxidase mutant, the encoding gene, the recombinant expression vector, and the recombinant cell.

[0021] NADPH oxidase mutants have important uses in the fields of pharmaceuticals, food, fine chemicals, pesticides, etc.

[0022] As a preferred embodiment of the present invention, the NADPH oxidase mutant, the encoding gene, the recombinant expression vector, and the recombinant cell are used to produce the coenzyme NADP+.

[0023] Specifically, the encoding gene, the recombinant expression vector, and the recombinant cell are used to produce NADPH oxidase, which can catalyze the oxidation reaction of NADPH to obtain coenzyme NADP+ and water.

[0024] Furthermore, the present invention also provides a method for producing coenzyme NADP+, comprising the following steps: taking NADPH and the NADPH oxidase mutant of the present invention, and adding them to a pH 7.0 sodium phosphate buffer.

[0025] Based on SmNOX, the present invention screened and obtained three SmNOX enzyme mutants through homology modeling technology and site-directed saturation mutagenesis technology. The catalytic activity of the SmNOX enzyme mutants was significantly improved, with a unit enzyme activity of 4.6 to 6.0 U / mg, which was 1.4 to 1.9 times higher than that of the original SmNOX enzyme. The SmNOX enzyme mutants can be more effectively used in related redox reactions and have good application prospects in the fields of pharmaceuticals, food, fine chemicals, pesticides, etc. DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] The experimental methods without specific conditions in the examples are all performed according to conventional molecular biology experimental methods, such as the methods described in Molecular Cloning Experimental Guide (J. Sambrook, DW Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, etc., third edition, Beijing: Science Press, 2002), or the methods recommended by the kit manufacturer.

[0028] Example 1 Construction of NADPH oxidase mutant expression vector

[0029] (I) Construction of SmNOX prokaryotic expression vector

[0030] The SmNOX gene fragment was synthesized by Changzhou Jiyu Biotechnology Co., Ltd. The gene sequence was codon-optimized according to the codon preference of Escherichia coli, and Nde I and Hind III restriction endonuclease sites were added at both ends of the coding region and recombined into the PUC57 vector.

[0031] The restriction endonucleases Nde I and Hind III (purchased from New England Biolabs, NEB) were double-digested at 37°C for 4 hours, and the digested products were separated by 1% agarose gel electrophoresis and recovered by gel excision (gel recovery kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The recovered products were connected with the pET28a(+) vector (Novagen) that had been double-digested in the same way, and the positive plasmid obtained by transformation and screening was transferred into the BL21(DE3) host bacteria to construct the in vitro heterologous expression system of SmNOX-pET28a(+) / BL21(DE3) expression strain.

[0032] (II) Site-directed mutagenesis of SmNOX prokaryotic expression vector to obtain NADPH oxidase mutant expression vector

[0033] Homology modeling technology was used to predict that the potentially beneficial mutation sites were 114, 116, and 118. Subsequently, the SmNOX-pET28a(+) recombinant plasmid was used as a template to perform site-directed saturation mutation of SmNOX at these three sites by PCR (for specific mutation operations, refer to the PCR product of Stratagene). Site-Directed Mutagenesis Kit Operating Instructions). The site-directed mutagenesis primers for each site are shown in Table 1 below.

[0034] Table 1 Site-directed mutagenesis primer sequences

[0035]

[0036] Example 2 Cultivation and Screening of Mutants (I) Preparation of Enzyme Supernatant by Shake Flask Fermentation

[0037] ① SmNOX enzyme solution: The expression strain SmNOX-pET28a(+) / BL21(DE3) constructed above was cultured in 5 mL LB liquid medium with a final concentration of 30 μg / mL kanamycin sulfate at 37°C and 200 rpm for overnight shaking, then inoculated into 500 mL LB liquid medium with a final concentration of 30 μg / mL kanamycin sulfate at a ratio of 1% (V / V), and cultured at 37°C and 200 rpm. When the absorbance OD600 was between 0.8-1.0, the inducer IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1 mM, and induced at 30°C overnight. The bacteria were collected by centrifugation at 4°C and 8000 rpm, then suspended in 50 mM pH 7.0 sodium phosphate buffer, ultrasonically disrupted (200 W, 3s / 6s, 20min), and centrifuged at 4°C and 12000 rpm for 20min. The supernatant was the enzyme solution.

[0038] LB liquid culture medium: 10 g / L tryptone (OXIOD), 5 g / L yeast powder (OXIOD), 10 g / L sodium chloride (Sinopharm Reagent).

[0039] ② Mutant enzyme solution: After the mutant plasmid obtained in Example 1 was transformed into the BL21 (DE3) host bacteria, it was spread on the LB solid medium containing 30 μg / mL kanamycin, and inverted and cultured at 37°C overnight. Then, a single clone was picked from the plate and placed in a 96-deep-well plate containing 1 mL LB liquid medium containing 30 μg / mL kanamycin sulfate for culture. The overnight cultured bacterial solution was then inoculated at a ratio of 1% (V / V) in a 96-deep-well plate containing 2 mL LB liquid medium containing 30 μg / mL kanamycin sulfate for induced expression. After shaking and culturing at 37°C and 200 rpm for 4 hours, IPTG with a final concentration of 0.1 mM was added for induction, and cultured at 25°C overnight.

[0040] Subsequently, the cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and after being thoroughly suspended by adding 2 mL of 50 mM pH 7.0 sodium phosphate buffer, lysozyme at a final concentration of 0.5 g / L was added and placed at 37°C for 3 h for cell wall disruption. Subsequently, the cells were centrifuged at 4°C and 4000 rpm for 20 min, and the supernatant was the enzyme solution of the mutants, which was used for mutant screening.

[0041] (II) Screening of mutants

[0042] Take NADPH and the enzyme solution prepared above and add them to 50mM pH7.0 sodium phosphate buffer, wherein the enzyme solution is added at a volume concentration of 1%, and the final concentration of NADPH is 0.2g / L. The absorbance changes of each enzyme solution are measured by an ELISA instrument to obtain the corresponding enzyme activity.

[0043] The method for determining enzyme activity is as follows: set the wavelength at 340nm, use the time scan mode to scan the change of absorbance within 5 minutes, record the absorbance values ​​before and after 5 minutes, and calculate the NADPH consumption rate through the NADPH standard curve.

[0044] The clones with significantly higher enzyme activity than the original SmNOX enzyme were expanded and sequenced to verify the mutation.

[0045] The sequencing results showed that the mutation sites in the clones with significantly improved mutant enzyme activity were as follows:

[0046] The threonine (T) at position 114 was mutated to glutamic acid (E), the isoleucine (I) at position 116 was mutated to phenylalanine (F), and the proline (P) at position 118 was mutated to serine (S).

[0047] The nucleotide sequences and amino acid sequences of the screened mutant enzymes are shown in Table 2 below.

[0048] Table 2 Nucleotide and amino acid sequences of each mutant enzyme

[0049] Mutation point situation Nucleotide sequence Amino acid sequence T114E SEQ ID No.3 SEQ ID No.4 I116F SEQ ID No.5 SEQ ID No.6 P118S SEQ ID No.7 SEQ ID No.8

[0050] Example 3 Mutant enzyme activity

[0051] The enzyme activities of the mutants were determined according to the method of Example 2. The results are shown in Table 3 below.

[0052] Table 3 Enzyme activities of various mutants

[0053] Mutants Unit enzyme activity U / mg Increase multiple Wild type NOX 3.2 -- T114E 6.0 1.9 I116F 5.5 1.7 P118S 4.6 1.4

[0054] *1U is defined as the amount of enzyme required to consume 1μmol NADPH in 1min.

[0055] As shown in Table 3, the enzyme activities of the mutants obtained by mutations T113E, I116F, and P118S were significantly improved, among which T114E had the most obvious improvement, with an increase of 1.9 times.

[0056] Finally, it should be noted that the above contents are only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Although the above embodiments describe the present invention in detail, those skilled in the art should understand that simple modification or equivalent replacement of the technical solution of the present invention without departing from the concept of the present invention does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A NADPH oxidase mutant, characterized in that The NADPH oxidase mutant is obtained by performing site-directed mutagenesis on the NADPH oxidase with an amino acid sequence as shown in SEQ ID NO:2, wherein the point mutation is T114E, I116F or P118S, and the corresponding amino acid sequence of the NADPH oxidase mutant is shown in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:

8.

2. A gene encoding the NADPH oxidase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO:

7.

4. A recombinant expression vector containing the coding gene as claimed in claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that The vector backbone of the recombinant expression vector includes one of pET, pCW, pUC and pPIC9k.

6. A recombinant cell comprising the coding gene according to claim 2 or 3.

7. The recombinant cell according to claim 6, characterized in that The host cell used in the recombinant cell includes one of Escherichia coli, Pichia pastoris, Streptomyces and Bacillus subtilis.

8. Use of the NADPH oxidase mutant according to claim 1, the encoding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or 5, and the recombinant cell according to claim 6 or 7 in producing the coenzyme NADP+.

9. A method for producing coenzyme NADP+, characterized in that: The method comprises the following steps: taking NADPH and the NADPH oxidase mutant according to claim 1, and adding them into a pH 7.0 sodium phosphate buffer.

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