CN113667651A

CN113667651A - NADH oxidase mutant with improved enzyme activity and changed optimal pH

Info

Publication number: CN113667651A
Application number: CN202110908307.1A
Authority: CN
Inventors: 杨套伟; 饶志明; 武文慧; 张显; 徐美娟; 邵明龙
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-19
Anticipated expiration: 2041-08-09
Also published as: CN113667651B

Abstract

The invention discloses an NADH oxidase mutant with improved enzyme activity and changed optimum pH, belonging to the technical field of genetic engineering. The NADH oxidase mutants N20D, Y66D, N116E, N20D/Y66D and N20D/N116E provided by the invention can achieve the technical effect of improving the enzyme activity under the neutral condition, in addition, the optimum pH value of the NADH oxidase mutant N20D/N116E is adjusted from 9.0 to 7.0, the specific enzyme activity is also obviously improved, the specific enzyme activity can reach 9.19U/mg protein at the pH value of 7.0 and is 2.9 times of the wild type, and the regeneration of NAD by the enzyme is enhanced⁺The capability of (2) provides a practical and effective coenzyme regeneration strategy for industrialized redox enzyme catalysis.

Description

NADH oxidase mutant with improved enzyme activity and changed optimal pH

Technical Field

The invention relates to an NADH oxidase mutant with improved enzyme activity and changed optimum pH, belonging to the technical field of genetic engineering.

Background

Industrial biocatalysis has become one of the emerging research directions with good development prospects in the field of biotechnology today. Enzymes play a critical role in industrial catalysis as an important catalyst. Statistically, only 15% of the enzymes that have been discovered and studied are industrially utilized, and among them, oxidoreductases, which are the largest class of enzymes, have important application values in the field of industrial biocatalysis. It is known that the vast majority of oxidoreductases require a coenzyme (NAD (P))⁺NAD (P) H) participates in the electron transfer to promote the conversion of the substrate. However, the coenzyme is expensive, unstable in solution and extremely low in recycling rate, and the addition of a large amount of coenzyme in a catalytic reaction system will greatly increase the reaction cost, which also results in severe limitation of further industrial application of the oxidoreductase. Therefore, the construction of a coenzyme regeneration system with high efficiency, environmental protection and low cost has important significance for the development of industrial biocatalysis, and the problems of high coenzyme cost, low utilization rate and the like in industrial application can be solved.

NADH oxidase (NOX enzyme) widely exists in various organisms, and can directly consume dissolved oxygen to catalyze and oxidize NADH to generate NAD⁺Thus, can be applied to NAD⁺Regeneration studies of (1). Producing H simultaneously₂The O-type NADH oxidase has the characteristic of high-efficiency catalysis, and has no byproduct generation, environmental friendliness, simple operation and great application potential in industry because the catalytic product is water. However, the optimal reaction pH of the current NADH oxidase is higher, the current NADH oxidase mostly depends on the reaction environment of strong alkali, and the enzyme activity is lower under the neutral condition, and the defect can definitely limit the application range of the NADH oxidase as a regeneration system.

Disclosure of Invention

In order to solve the technical problems that the optimal reaction pH of NADH oxidase is higher, the NADH oxidase mostly depends on the reaction environment of strong alkali, the enzyme activity is lower under the neutral condition and the like in the prior art, the invention provides an NADH oxidase mutant, the enzyme activity of the NADH oxidase mutant is improved under the mild condition, and the NAD oxidase mutant can be used for improving the NAD⁺The reaction efficiency of the regeneration system is improved, and the reaction cost is reduced.

The mutant is obtained by mutating asparagine at the 20 th site of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid; named N20D;

or the mutant is obtained by mutating tyrosine at position 66 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into aspartic acid; named Y66D;

or the mutant is obtained by mutating asparagine at the 116 th site of NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 into glutamic acid; named N116E;

or the mutant is obtained by mutating asparagine at the 20 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid and simultaneously mutating tyrosine at the 66 th position into aspartic acid; named N20D/Y66D; the amino acid sequence of the mutant is shown as SEQ ID NO. 3;

or the mutant is obtained by mutating asparagine at the 20 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid and simultaneously mutating asparagine at the 116 th position into glutamic acid; named N20D/N116E; the amino acid sequence of the mutant is shown in SEQ ID NO. 4.

In one embodiment of the invention, the nucleotide sequence encoding said NADH oxidase is shown in SEQ ID NO. 2.

The invention also provides a gene for coding the NADH oxidase mutant.

The invention also provides a recombinant vector carrying the gene.

In one embodiment of the invention, the recombinant vector uses pETDust series vectors as starting vectors.

In one embodiment of the invention, the recombinant vector uses pETDuet-1 as a starting vector.

The invention also provides a recombinant cell expressing the mutant, or carrying the gene, or carrying the recombinant vector.

In one embodiment of the present invention, the recombinant cell has Escherichia coli as a host cell.

In one embodiment of the invention, the recombinant cell is a host cell of Escherichia coli BL21(DE 3).

The invention also provides a method for reducing the most suitable pH of NADH oxidase and improving enzyme activity,

mutating asparagine at position 20 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into aspartic acid;

or mutating tyrosine at position 66 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 to aspartic acid;

or the asparagine at the 116 th site of the NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 is mutated into glutamic acid;

or the mutant is obtained by mutating asparagine at position 20 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into aspartic acid, and simultaneously mutating tyrosine at position 66 into aspartic acid;

or the mutant is obtained by mutating asparagine at position 20 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into aspartic acid, and simultaneously mutating asparagine at position 116 into glutamic acid.

The invention also provides a regenerated coenzyme NAD⁺The method of (1), wherein the mutant or the recombinant cell is added to a reaction system containing dissolved oxygen and NADH, and NAD is prepared by reaction⁺。

In one embodiment of the present invention, the reaction conditions are: 20 to 28 ℃ and pH7.0 to 7.5.

In one embodiment of the present invention, the reaction conditions are: at 25 ℃ and pH 7.0.

In one embodiment of the present invention, the concentration of dissolved oxygen in the reaction system is: 3-3.5 mg/L.

In one embodiment of the present invention, the concentration of NADH in the reaction system is: 0.2 to 1 mM.

In one embodiment of the invention, the regenerated NAD provided herein⁺The method of (1), wherein the microbial cell or mutant is added without any side substrate and no by-product is generatedUnder the condition of regenerating NADH into NAD⁺。

In one embodiment of the invention, the pH optimum of NADH oxidase mutant N20D/N116E is reduced from 9.0 in the wild type to 7.0, while the specific enzyme activity at pH7.0 is 2.9 times that of the wild type.

The invention also provides the mutant, the gene, the recombinant plasmid or the recombinant cell for increasing intracellular NAD (nicotinamide adenine dinucleotide) of the microorganism⁺Content of or in preparation of the composition can increase intracellular NAD of microorganisms⁺The application in the product with the content.

Advantageous effects

(1) The invention adopts H₂The O-type NADH oxidase has the characteristic of high-efficiency catalysis, no byproduct is generated due to the fact that a catalytic product of the oxidase is water, the O-type NADH oxidase is environment-friendly and simple to operate, the optimum pH value of the NADH oxidase mutant provided by the invention is neutral, the defects that the NADH oxidase in the prior art depends on a strong alkali reaction environment and the enzyme activity is low under a neutral condition are overcome, and the O-type NADH oxidase has great application potential in industry.

(2) The NADH oxidase mutants N20D, Y66D, N116E, N20D/Y66D and N20D/N116E provided by the invention can achieve the technical effect of improving the enzyme activity under the neutral condition, in addition, the optimum pH value of the NADH oxidase mutant N20D/N116E is adjusted to 7.0 from 9.0, the specific enzyme activity is also obviously improved, the specific enzyme activity can reach 9.19U/mg protein at the pH value of 7.0 and is 2.9 times of the wild type, the capacity of the enzyme for regenerating NAD + is enhanced, and a practical and effective coenzyme regeneration strategy is provided for the industrial oxidoreductase catalysis.

Drawings

FIG. 1: the single-site mutant and the wild type have specific enzyme activities at different pH values.

FIG. 2: the specific enzyme activities of the double-site mutant and the wild type are at different pH values.

FIG. 3: agarose gel electrophoresis of crude enzyme solutions containing wild type and mutant NADH oxidase; wherein, lane 1 protein maker, lane 2: the blank crude enzyme solution,

lanes

3 and 4 are the precipitate and supernatant of wild-type crude enzyme solution of NADH oxidase,

lanes

5 and 6 are the precipitate and supernatant of crude enzyme solution of mutant N20D,

lanes

7 and 8 are the precipitate and supernatant of crude enzyme solution of mutant N116E,

lanes

9 and 10 are the precipitate and supernatant of crude enzyme solution of mutant N20D/N116E, and

lanes

11 and 12 are the precipitate and supernatant of crude enzyme solution of mutant N20D/N116E.

FIG. 4: agarose gel electrophoresis images of pure enzyme solutions of wild type and mutant NADH oxidase; the marker in lane 1, other null proteins in

lanes

2 and 3, and the primase, mutant N20D, mutant N116E, mutant N20D/N116E, and mutant Y119E in

lanes

5, 6, 7, 8, and 9, respectively.

Detailed Description

The media involved in the following examples are as follows:

LB liquid medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH 7.2.

LB solid medium: 20g/L agar was added to the LB liquid medium.

The detection methods referred to in the following examples are as follows:

the method for measuring the enzymatic activity of the NADH oxidase comprises the following steps:

reaction system: (1.0mL system) 50mm potassium phosphate, 0.3mm EDTA, 50 μm FAD, 0.3mm β -NADH, 10 μ L enzyme solution. Reaction conditions are as follows: at 25 ℃ and pH 7.0. The NOX enzyme activity was determined spectrophotometrically by monitoring the change in NADH absorbance at 340nm, three replicates.

… formula: …

Δ A: amount of change in absorbance

… 1: reacting for one minute

… … … … … … 0.5.5: depending on the type of cuvette, others are constants.

Definition of enzyme activity: the amount of enzyme required to consume or produce 1. mu. mol NADH per minute is defined as one enzyme activity unit U.

Specific enzyme activity definition: the unit protein enzyme activity U/mg.

The purification methods referred to in the following examples are as follows:

the eluents required were as follows:

m0: 20mM Tris-HCl (pH8.0), 500mM NaCl, 10% glycerol;

m1000: 20mM Tris-HCl (pH8.0), 500mM NaCl, 10% glycerol, 1M imidazole;

mixing M0 and M1000 according to a certain proportion to obtain gradient eluent: m50, M80, M100, M200 and M300, which are eluents with imidazole concentrations of 50mM, 80mM, 100mM, 200mM and 300mM, respectively.

The purification process was as follows: washing the chromatographic column with 2 times of column volume of ultrapure water and M0 solution, filtering the crude enzyme solution with 0.45 μ M microfiltration membrane, purifying with the recombinant protein and Ni on the chromatographic column⁺And (4) combining. After the crude enzyme solution is applied to the column, M0 solution with the volume 10-20 times of the column volume is used for eluting the non-combined hybrid protein, then M50 with the volume 2 times of the column volume, M80, M100 and M200 solution with the volume 1 time of the column volume are used for washing the column in sequence, the hybrid protein which is not combined with Ni + in a specific way is washed, finally M300 solution is used for eluting the target protein, and the eluent is collected by tubes.

The mutant primers referred to in the following examples are as follows:

TABLE 1 primers required for PCR

Example 1: construction of recombinant bacterium containing gene encoding NADH oxidase mutant

The method comprises the following specific steps:

(1) chemically synthesizing NADH oxidase WT with the nucleotide sequence shown in SEQ ID NO.2, carrying out enzyme digestion on the target gene fragment and the plasmid pETDuet-1 by utilizing restriction enzymes Nde I and Xho I, and then connecting to prepare the recombinant plasmid pETDuet-1-WT containing the NADH oxidase WT.

(2) Constructing mutant plasmids by using pETDuet-1-WT as a template and a PCR method according to a primer sequence shown in table 1 (a reaction system is shown in table 2, and reaction conditions are shown in table 3), wherein double mutation is constructed and prepared by using a corresponding primer sequence on the basis of single mutation; namely, T2D, N20D, K25D, Y66E, Y66D, Q68E, K85D, N116E, Q114E, A118D, Y119E, I168D, R171E, H188E, Q189D, K197D, T2D/N20D, T2D/K25D, T2D/N116E, T2D/A118D, N20D/Y66D, N20D/N116E, N20D/A118D, K25D/N116E, K25D/A118D are obtained.

TABLE 2 PCR reaction System

TABLE 3 PCR reaction conditions

(3) The PCR products were examined by gel electrophoresis, and then 20. mu.L of the PCR products were digested with 1. mu.L of Dpn I restriction enzyme, and incubated at 37 ℃ for 1 hour.

Absorbing 5 mu L of enzyme digestion products and transforming the enzyme digestion products into escherichia coli BL21(DE3) to obtain corresponding recombinant escherichia coli, coating the recombinant escherichia coli on an LB plate containing ampicillin (100mg/L), culturing overnight at 37 ℃, randomly selecting clones to perform colony PCR identification and sequencing verification, and the result shows that the recombinant expression vector containing the gene for encoding the NADH oxidase mutant is successfully transformed into an expression host escherichia coli BL21(DE 3); the bacterial strain which is successfully mutated through sequencing verification is the recombinant bacterial strain containing the mutant and is respectively named as:

E.coli BL21/pETDuet-1-WT，E.coli BL21/pETDuet-1-T2D，E.coli BL21/pETDuet-1-N20D，E.coli BL21/pETDuet-1-K25D，E.coli BL21/pETDuet-1-Y66E，E.coli BL21/pETDuet-1-Y66D，E.coli BL21/pETDuet-1-Q68E，E.coli BL21/pETDuet-1-K85D，E.coli BL21/pETDuet-1-N116E，E.coli BL21/pETDuet-1-Q114E，E.coli BL21/pETDuet-1-A118D，E.coli BL21/pETDuet-1-Y119E，E.coli BL21/pETDuet-1-I168D，E.coli BL21/pETDuet-1-R171E，E.coli BL21/pETDuet-1-H188E，E.coli BL21/pETDuet-1-Q189D，E.coli BL21/pETDuet-1-K197D，E.coli BL21/pETDuet-1-T2D/N20D，E.coli BL21/pETDuet-1-T2D/K25D，E.coli BL21/pETDuet-1-T2D/N116E，E.coli BL21/pETDuet-1-T2D/A118D，E.coli BL21/pETDuet-1-N20D/Y66D，E.coli BL21/pETDuet-1-N20D/N116E，E.coli BL21/pETDuet-1-N20D/A118D，E.coli BL21/pETDuet-1-K25D/N116E，E.coli BL21/pETDuet-1-K25D/A118D。

adding the recombinant strain into glycerol and preserving in a refrigerator at the temperature of-70 ℃; wherein, the sequencing work is completed by the Jinzhi Zhi of Suzhou.

Example 2: expression of NADH oxidase mutant

The method comprises the following specific steps:

(1) the recombinant bacteria constructed in example 1 were respectively inoculated into 10mL LB liquid medium containing ampicillin, cultured with shaking at 37 ℃ for 12h, and cultured to OD₆₀₀0.6-0.9, and respectively preparing seed solutions;

(2) the seed liquid is respectively transferred into 50mL LB liquid culture medium containing 100ug/mL ampicillin according to the inoculation amount of 1% (v/v), cultured for 2-3 h at 37 ℃, added with 0.5mM IPTG, and induced for 12-16 h at 16 ℃, and then respectively obtained fermentation liquid.

(3) The fermentation liquid was centrifuged at 8000rpm and 4 ℃ for 10min, cells were collected and disrupted, cell disruption supernatant (crude enzyme solution) was collected for subsequent purification, and a portion of the crude enzyme solution was subjected to agarose gel electrophoresis analysis, the results of which are shown in FIG. 3.

(4) And (4) purifying the crude enzyme solution prepared in the step (3) to prepare purified enzyme, and storing the purified enzyme at 4 ℃ for later use.

The purified enzyme solution was analyzed by SDS-PAGE, and the results are shown in FIG. 4 (electrophoresis pattern of only a part of mutants is shown due to the large number of mutants), which shows that electrophoretically pure recombinant NADH oxidase and its mutants were obtained, namely: pure enzyme solution containing WT, pure enzyme solution containing T2, pure enzyme solution containing N20, pure enzyme solution containing K25, pure enzyme solution containing Y66, pure enzyme solution containing Q68, pure enzyme solution containing K85, pure enzyme solution containing N116, pure enzyme solution containing Q114, pure enzyme solution containing A118, pure enzyme solution containing Y119, pure enzyme solution containing I168, pure enzyme solution containing R171, pure enzyme solution containing H188, pure enzyme solution containing Q189, pure enzyme solution containing K197, pure enzyme solution containing T2/N20, pure enzyme solution containing T2/K25, pure enzyme solution containing T2/N116, pure enzyme solution containing T2/A118, pure enzyme solution containing N20/Y66, pure enzyme solution containing N20/N116, pure enzyme solution containing N20/A118, pure enzyme solution containing K25/N116, and pure enzyme solution containing K118/K25/A118.

And (3) taking the no-load plasmid as a blank control, and respectively preparing a blank crude enzyme solution and a blank pure enzyme solution according to the method.

Example 3: enzymatic activity determination of NADH oxidase

The method comprises the following specific steps:

the pure enzyme solution prepared in example 2 was used to adjust the pH value in the enzyme activation reaction system at 25 ℃ by means of dipotassium hydrogen phosphate/potassium dihydrogen phosphate, i.e. the specific activities of the NADH oxidase primary enzyme and the mutant at pH7.0, pH8.0, and pH 9.0 were measured, and the results are shown in table 4 and fig. 1-2; in FIG. 2, T2D + N20D, T2D + K25D, T2D + N116E, T2D + A118D, N20D + Y66D, N20D + N116E, N20D + A118D, K25D + N116E and K25D + A118D represent T2D/N20D, T2D/K25D, T2D/N116E, T2D/A118D, N20D/Y66D, N20D/N116E, N20D/A118D, K25D/N116E and K25D/A118D mutants in abscissa.

Because mutation data is more, specific enzyme activity data can be obtained in fig. 1-2, and therefore, only listed in table 4: original enzyme, single mutation sites N20D, Y66E, Y66D, N116E and Y119E, and double mutation sites T2D/N20D, T2D/K25D, T2D/N116E, N20D/Y66D and N20D/N116E. The activity of NADH oxidase NOX was determined by monitoring the change in NADH absorbance at 340 nm.

Table 4: specific enzyme activity (U/mg) of original enzyme and mutant enzyme at different pH

The results show that the mutation of the Y119E mutant is the least favorable of all the mutations, wherein the enzyme activity is completely lost at pH7.0, and the enzyme activities at pH8.0 and 9.0 are respectively reduced by 99.98% and 99.88% compared with the original enzyme, so that the site is not adopted in subsequent experiments.

Taking the mutant N20D/N116E as an example, the mutant enables pH7.0 to be the optimum reaction pH, meanwhile, the specific enzyme activity of the pH mutant strain is 9.2U/mg, which is 2.9 times of the original enzyme, and the specific enzyme activity of the enzyme under neutral conditions (pH 7.0) is obviously improved through multi-site mutation. The specific enzyme activity of the mutant N20D/N116E at pH8.0 was 2.2 times that of the original enzyme.

It can be seen that the mutant N20D/N116E has enzyme activity improved to different degrees under different pH conditions, and the optimum pH is reduced to pH7.0, which means that the mutant of the invention can be applied to the enzyme catalysis reaction with mild conditions, and can effectively improve NAD⁺The regeneration capability of the method provides a practical and effective coenzyme regeneration strategy for industrialized redox enzyme catalysis.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. An NADH oxidase mutant, characterized in that the mutant is obtained by mutating asparagine at position 20 of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid;

or the mutant is obtained by mutating tyrosine at position 66 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into aspartic acid;

or the mutant is obtained by mutating asparagine at the 116 th site of NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 into glutamic acid;

or the mutant is obtained by mutating asparagine at the 20 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid and simultaneously mutating tyrosine at the 66 th position into aspartic acid;

or the mutant is obtained by mutating asparagine at the 20 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid and simultaneously mutating asparagine at the 116 th position into glutamic acid.

2. A gene encoding said mutant NADH oxidase of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, wherein the recombinant plasmid uses pETDust series vector as starting vector.

5. A recombinant cell expressing the mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3 or 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is E.coli as a host cell.

7. A method for reducing the optimum pH of NADH oxidase and increasing the enzyme activity, characterized in that,

or obtained by mutating asparagine at position 116 of NADH oxidase with amino acid sequence shown as SEQ ID NO.1 into glutamic acid;

8. Regenerated coenzyme NAD⁺The method of (1), or the recombinant cell of claim 5 or 6, is added to a reaction system containing dissolved oxygen and NADH, and NAD is produced by the reaction⁺。

9. The method of claim 8, wherein the reaction conditions are: 20 to 28 ℃ and pH7.0 to 7.5.

10. The mutant of claim 1, or the gene of claim 2, or the recombinant plasmid of claim 3 or 4, or the recombinant of claim 5 or 6Increasing intracellular NAD of microorganisms by cells⁺Content of or in preparation of the composition can increase intracellular NAD of microorganisms⁺The application in the product with the content.