CN113667651B - NADH oxidase mutant with improved enzyme activity and changed optimal pH - Google Patents
NADH oxidase mutant with improved enzyme activity and changed optimal pH Download PDFInfo
- Publication number
- CN113667651B CN113667651B CN202110908307.1A CN202110908307A CN113667651B CN 113667651 B CN113667651 B CN 113667651B CN 202110908307 A CN202110908307 A CN 202110908307A CN 113667651 B CN113667651 B CN 113667651B
- Authority
- CN
- China
- Prior art keywords
- nadh oxidase
- mutant
- ala
- enzyme activity
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
- C12Y106/03001—NAD(P)H oxidase (1.6.3.1), i.e. NOX1
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an NADH oxidase mutant with improved enzyme activity and changed optimal pH value, belonging to the technical field of genetic engineering. The NADH oxidase mutant N20D, Y66D, N116E, N D/Y66D, N20D/N116E provided by the invention can realize the technical effect of improving the enzyme activity under neutral conditions, the optimal pH value of the NADH oxidase mutant N20D/N116E is reduced 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 the specific enzyme activity is 2.9 times that of the wild type, thus strengthening the regeneration of NAD by the enzyme + Provides a practical and effective coenzyme regeneration strategy for the catalysis of industrial oxidoreductase.
Description
Technical Field
The invention relates to an NADH oxidase mutant with improved enzyme activity and changed optimal 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 biotechnology field. Enzymes act as an important catalyst and play a critical role in industrial catalysis. Only 15% of enzymes found and studied have been statistically used in industry, with oxidoreductases as the largest class of enzymes, with important application value in the field of industrial biocatalysis. It is known that the vast majority of oxidoreductases require coenzyme (NAD (P) in the catalytic process + NAD (P) H is involved in electron transfer to facilitate substrate conversion. However, the cost of the coenzyme is high, the coenzyme is unstable in the solution, the recycling rate is extremely low, and the addition of a large amount of the coenzyme in the catalytic reaction system greatly increases the reaction cost, which also results in serious limitation of further industrial application of the oxidoreductase. Therefore, the construction of efficient, environment-friendly and low-cost coenzyme regeneration systems is important for the development of industrial biocatalysisMeaning, can also solve the difficult problems of high coenzyme cost, low utilization rate and the like in industrial application.
NADH oxidase (NOX enzyme) is widely available in various organisms and can directly consume dissolved oxygen to catalyze and oxidize NADH to generate NAD + Therefore, can be applied to NAD + Is a regeneration study of (a). Simultaneous H production 2 The O-type NADH oxidase has the characteristics of high efficiency catalysis, and has the advantages of no byproduct generation, environmental friendliness and simple operation because the catalysis product is water, and has great application potential in industry. However, the optimal reaction pH of the current NADH oxidase is high, the optimal reaction pH depends on the reaction environment of strong alkali, the enzyme activity is low under neutral conditions, 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 in the prior art, the optimal reaction pH of NADH oxidase is higher, the reaction environment of strong base is relied on, the enzyme activity is lower under neutral conditions, and the like, the invention provides an NADH oxidase mutant, and the enzyme activity of the NADH oxidase mutant is improved under mild conditions, so that NAD can be improved + The reaction efficiency of the regeneration system is reduced, and the reaction cost is reduced.
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; designated as N20D;
or the mutant is obtained by mutating tyrosine at 66 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid; designated Y66D;
or the mutant is obtained by mutating asparagine at position 116 of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into glutamic acid; designated 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 mutating tyrosine at the 66 th position into aspartic acid; named as N20D/Y66D; the amino acid sequence of the mutant is shown as SEQ ID NO. 3;
or the mutant is obtained by mutating the 20 th asparagine of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 into aspartic acid, and mutating the 116 th asparagine into glutamic acid; named N20D/N116E; the amino acid sequence of the mutant is shown as SEQ ID NO. 4.
In one embodiment of the invention, the nucleotide sequence encoding the NADH oxidase is shown in SEQ ID NO. 2.
The invention also provides a gene for encoding 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 present invention, the recombinant vector uses pETDuet-1 as a starting vector.
The invention also provides a recombinant cell for expressing the mutant, carrying the gene or carrying the recombinant vector.
In one embodiment of the invention, the recombinant cell uses E.coli as a host cell.
In one embodiment of the invention, the recombinant cell is a host cell of E.coli BL21 (DE 3).
The invention also provides a method for reducing the optimal pH value of NADH oxidase and improving the enzyme activity,
mutating asparagine at position 20 of NADH oxidase with amino acid sequence shown in SEQ ID NO.1 into aspartic acid;
or mutating the tyrosine at the 66 th position of NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 into aspartic acid;
or mutating the 116 th asparagine of NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 into glutamic acid;
or the mutant is characterized in that asparagine at the 20 th position of NADH oxidase with an amino acid sequence shown as SEQ ID NO.1 is mutated into aspartic acid, and tyrosine at the 66 th position is mutated into aspartic acid;
or the mutant is characterized in that the asparagine at the 20 th position of NADH oxidase with the amino acid sequence shown as SEQ ID NO.1 is mutated into aspartic acid, and the asparagine at the 116 th position is mutated into glutamic acid.
The invention also provides a regenerated coenzyme NAD + The method comprises adding the mutant or the recombinant cell into a reaction system containing dissolved oxygen and NADH, and reacting to obtain NAD + 。
In one embodiment of the invention, the reaction conditions are: 20-28 ℃ and pH 7.0-7.5.
In one embodiment of the invention, the reaction conditions are: 25℃and pH7.0.
In one embodiment of the present invention, the concentration of dissolved oxygen in the reaction system is: 3-3.5mg/L.
In one embodiment of the invention, the concentration of NADH in the reaction system is: 0.2 to 1mM.
In one embodiment of the invention, the invention provides a regenerated NAD + In the presence of no by-product, NADH is regenerated into NAD by using the microbial cells or mutants without the addition of a by-substrate + 。
In one embodiment of the invention, the pH optimum of the NADH oxidase mutant N20D/N116E is reduced from 9.0 of 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 of the microorganism + Content, or in the preparation of intracellular NAD increasing the microorganism + The application of the content in the product.
Advantageous effects
(1) The invention adopts H 2 The O-type NADH oxidase has the characteristics of high efficiency catalysis, has no byproduct generation because the catalysis product is water, is environment-friendly and simple to operate, and the optimal pH value of the NADH oxidase mutant provided by the invention is neutral, thereby solving the problems that the NADH oxidase in the prior art depends on the reaction environment of strong alkaliThe defect of low enzyme activity under neutral conditions has great application potential in industry.
(2) The NADH oxidase mutant N20D, Y66D, N116E, N D/Y66D, N20D/N116E provided by the invention can realize the technical effect of improving the enzyme activity under neutral conditions, the optimal pH of the NADH oxidase mutant N20D/N116E is reduced from 9.0 to 7.0, the specific enzyme activity is obviously improved, the specific enzyme activity can reach 9.19U/mg protein at the pH of 7.0, the specific enzyme activity is 2.9 times that of a wild type, the NAD+ regeneration capability of the enzyme is enhanced, and an actual effective coenzyme regeneration strategy is provided for the catalysis of industrial oxidoreductase.
Drawings
Fig. 1: specific enzyme activities of single-site mutants and wild-type at different pH.
Fig. 2: specific enzyme activities of the double-site mutant and the wild type at different pH values.
Fig. 3: agarose gel electrophoresis of crude enzyme solutions containing NADH oxidase wild-type and mutant; wherein, lane 1 protein maker, lane 2: blank crude enzyme solution, lanes 3 and 4 are respectively the precipitate and supernatant of NADH oxidase wild type crude enzyme solution, lanes 5 and 6 are respectively the precipitate and supernatant of mutant N20D crude enzyme solution, lanes 7 and 8 are respectively the precipitate and supernatant of mutant N116E crude enzyme solution, lanes 9 and 10 are respectively the precipitate and supernatant of mutant N20D/N116E crude enzyme solution, and lanes 11 and 12 are respectively the precipitate and supernatant of mutant N20D/N116E crude enzyme solution.
Fig. 4: agarose gel electrophoresis of pure enzyme solutions of NADH oxidase wild-type and mutant; lanes 1, protein maker, 2,3 and other inactive proteins, lanes 5,6,7,8,9 are primordial enzyme, mutant N20D, mutant N116E, mutant N20D/N116E, mutant Y119E, respectively.
Detailed Description
The following examples relate to the following media:
LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2.
LB solid medium: 20g/L agar was added on the basis of LB liquid medium.
The detection method involved in the following examples is as follows:
the method for measuring the enzyme activity of NADH oxidase comprises the following steps:
the reaction system: (1.0 mL system) 50mm potassium phosphate, 0.3mm EDTA, 50 μm FAD,0.3mm beta-NADH, 10. Mu.L enzyme solution. Reaction conditions: 25℃and pH7.0. Spectrophotometry measures NOX enzyme activity by monitoring the change in absorbance of NADH at 340nm, three in parallel.
… the formula: …
Δa: variation of absorbance
… 1: reaction for one minute
… … … … … … 0.5.5: the others are constant, depending on the type of cuvette.
Definition of enzyme activity: the amount of enzyme required to consume or generate 1. Mu. Mol NADH per minute is defined as one enzyme activity unit U.
Specific enzyme activity definition: u/mg enzyme activity per protein.
The purification methods involved in the following examples are as follows:
the required eluents were as follows:
m0:20mM Tris-HCl (pH 8.0), 500mM NaCl,10% glycerol;
m1000:20mM Tris-HCl (pH 8.0), 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 are eluents with imidazole concentrations of 50mM, 80mM, 100mM, 200mM and 300mM, respectively.
The purification process is as follows: washing chromatographic column with 2 times of column volume of ultrapure water and M0 solution, filtering crude enzyme solution with micro-filtration membrane with aperture of 0.45 μm, purifying with column, and purifying recombinant protein and Ni on chromatographic column + And (5) combining. After the crude enzyme solution is loaded on the column, firstly eluting unbound impurity protein by using 10-20 times of M0 solution with the column volume, then sequentially flushing the column by using 2 times of M50,1 time of M80, M100 and M200 solutions with the column volume respectively, washing impurity protein which is non-specifically bound with Ni+ and finally using M300 solutionEluting target protein, and collecting eluent by branch pipes.
The following examples refer to the mutation primers 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) The NADH oxidase WT with the nucleotide sequence shown in SEQ ID NO.2 is chemically synthesized, the target gene fragment is subjected to enzyme digestion with plasmid pETDuet-1 by utilizing restriction endonucleases Nde I and Xho I, and then the recombinant plasmid pETDuet-1-WT containing the NADH oxidase WT is prepared by connecting.
(2) Constructing mutant plasmids (a reaction system is shown in a table 2, and reaction conditions are shown in a table 3) by using pETDuet-1-WT as a template and adopting a PCR method according to a primer sequence shown in the table 1, wherein double mutation is prepared by constructing a corresponding primer sequence on the basis of single mutation; 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 checked by gel electrophoresis, and 1. Mu.L of Dpn I restriction enzyme was added to 20. Mu.L of the PCR products to digest the template plasmid, and incubated at 37℃for 1 hour.
Absorbing 5 mu L of enzyme digestion products, converting into escherichia coli BL21 (DE 3) to obtain corresponding recombinant escherichia coli, coating the corresponding recombinant escherichia coli on an ampicillin (100 mg/L) LB plate, culturing overnight at 37 ℃, randomly picking clones for colony PCR identification and sequencing verification, and successfully converting a recombinant expression vector containing a gene for encoding an NADH oxidase mutant into an expression host escherichia coli BL21 (DE 3); the strains which are successfully mutated by sequencing verification are recombinant strains containing mutants, and are respectively named as follows:
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 glycerol into the recombinant bacteria and preserving at-70 ℃; among them, sequencing work was done by su state Jin Weizhi.
Example 2: expression of NADH oxidase mutants
The method comprises the following specific steps:
(1) Inoculating the recombinant bacteria constructed in example 1 into 10mL LB liquid medium containing ampicillin, shake culturing at 37deg.C for 12 hr, and culturing to OD 600 0.6 to 0.9, respectively preparing seed liquid;
(2) The seed solutions are respectively transferred into 50mL of LB liquid medium containing 100ug/mL of ampicillin according to the inoculum size of 1% (v/v), cultured for 2-3 h at 37 ℃, added with 0.5mM IPTG, and induced for 12-16 h at 16 ℃ to obtain fermentation broths respectively.
(3) After the above fermentation broths were centrifuged at 8000rpm at 4℃for 10min, cells were collected and disrupted, and cell disruption supernatant (crude enzyme liquid) was collected for subsequent purification, and the resulting crude enzyme liquid was partially analyzed by agarose gel electrophoresis, and the results are shown in FIG. 3.
(4) Purifying the crude enzyme solution prepared in the step (3) to prepare purified enzyme, and storing the obtained purified enzyme at 4 ℃ for later use.
The purified enzyme solution was analyzed by SDS-PAGE, and the results are shown in FIG. 4 (only the electropherograms of some mutants are listed because of the large number of mutants), and the results indicate that the electrophoretically pure recombinant NADH oxidase and the mutants thereof are obtained, namely, respectively: pure enzyme solutions containing WT, pure enzyme solution containing N20D, pure enzyme solution containing K25D, pure enzyme solution containing Y66E, pure enzyme solution containing Y66D, pure enzyme solution containing Q68E, pure enzyme solution containing K85D, pure enzyme solution containing N116E, pure enzyme solution containing Q114E, pure enzyme solution containing A118D, pure enzyme solution containing Y119E, pure enzyme solution containing I168D, pure enzyme solution containing R171E, pure enzyme solution containing H188E, pure enzyme solution containing Q189D, pure enzyme solution containing K197D, pure enzyme solution containing T2D/N20D, pure enzyme solution containing T2D/K25D, pure enzyme solution containing T2D/N116E, pure enzyme solution containing T2D/A118D, pure enzyme solution containing N20D/Y66D, pure enzyme solution containing N20D/N116E, pure enzyme solution containing N20D/N25D, pure enzyme solution containing K118D and pure enzyme solution containing K25D/118D.
And taking the empty plasmid as a blank control, and respectively preparing blank crude enzyme liquid and blank pure enzyme liquid 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 the example 2 is respectively regulated to pH value in an enzyme activity reaction system by dipotassium hydrogen phosphate/potassium dihydrogen phosphate at the temperature of 25 ℃, namely, the specific enzyme activities of NADH oxidase original enzyme and mutant under the conditions of pH7.0, pH8.0 and pH 9.0 are respectively detected, and the results are shown in Table 4 and figures 1-2; in FIG. 2, T2D+N20D, T2D+K25D, T2D+N116E, T2D+A118D, N D+Y66D, N20D+N116E, N20D+A118D, K25D+N116E, K25D+A118D represents T2D/N20D, T2D/K25D, T D/N116E, T2D/A118D, N20D/Y66D, N20D/N116E, N20D/A118D, K3525D/N116E, K D/A118D mutant, respectively.
Since mutation data are more, specific enzyme activity data can be obtained in fig. 1-2, and therefore are listed only in table 4: specific enzyme activity data for the original enzyme, single mutation site N20D, Y66E, Y D, N35116E, Y E, and double mutation site T2D/N20D, T2D/K25D, T D/N116E, N20D/Y66D, N D/N116E. The activity of NADH oxidase NOx was determined by monitoring the change in absorbance of NADH at 340 nm.
Table 4: specific enzyme activities (U/mg) of the original and mutant enzymes at different pH values
The results showed that the mutation results of the Y119E mutant were the worst among all the mutations, in which the enzyme activity was completely lost at pH7.0, and the enzyme activities at pH8.0 and 9.0 were reduced by 99.98%,99.88%, respectively, compared to the original enzyme, so that this site was not used in the subsequent experiments.
Taking an N20D/N116E mutant as an example, the mutant enables pH7.0 to be the optimal reaction pH, meanwhile, the specific enzyme activity of the pH mutant is 9.2U/mg, which is 2.9 times of that of the original enzyme, and the specific enzyme activity of the enzyme under neutral (pH 7.0) is obviously improved by multi-site mutation. At pH8.0, the specific enzyme activity of the mutant N20D/N116E was 2.2 times that of the original enzyme.
The mutant N20D/N116E can be used for improving the NAD more effectively under the condition of different pH values, and the optimal pH value is adjusted to be 7.0 + Provides a practical and effective coenzyme regeneration strategy for the catalysis of industrial oxidoreductase.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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
<110> university of Jiangnan
<120> an NADH oxidase mutant with improved enzymatic activity and modified pH optimum
<130> BAA211018A
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 202
<212> PRT
<213> artificial sequence
<400> 1
Met Thr Asn Thr Leu Asp Val Leu Lys Ala Arg Ala Ser Val Lys Glu
1 5 10 15
Tyr Asp Thr Asn Ala Pro Ile Ser Lys Glu Glu Leu Thr Glu Leu Leu
20 25 30
Asp Leu Ala Thr Lys Ala Pro Ser Ala Trp Asn Leu Gln His Trp His
35 40 45
Phe Thr Val Phe His Ser Asp Glu Ser Lys Ala Glu Leu Leu Pro Val
50 55 60
Ala Tyr Asn Gln Lys Gln Ile Val Glu Ser Ser Ala Val Val Ala Ile
65 70 75 80
Leu Gly Asp Leu Lys Ala Asn Glu Asn Gly Glu Glu Val Tyr Ala Glu
85 90 95
Leu Ala Ser Gln Gly Tyr Ile Thr Asp Glu Ile Lys Gln Thr Leu Leu
100 105 110
Gly Gln Ile Asn Gly Ala Tyr Gln Ser Glu Gln Phe Ala Arg Asp Ser
115 120 125
Ala Phe Leu Asn Ala Ser Leu Ala Ala Met Gln Leu Met Ile Ala Ala
130 135 140
Lys Ala Lys Gly Tyr Asp Thr Cys Ala Ile Gly Gly Phe Asn Lys Glu
145 150 155 160
Gln Phe Gln Lys Gln Phe Asp Ile Ser Glu Arg Tyr Val Pro Val Met
165 170 175
Leu Ile Ser Ile Gly Lys Ala Val Lys Pro Ala His Gln Ser Asn Arg
180 185 190
Leu Pro Leu Ser Lys Val Ser Thr Trp Leu
195 200
<210> 2
<211> 609
<212> DNA
<213> artificial sequence
<400> 2
atgacgaata ctctggatgt tttaaaagca cgtgcatctg taaaggaata tgatacaaat 60
gccccgatct ctaaggagga gctgactgag ctattagacc ttgccactaa agcgccttct 120
gcttggaacc ttcagcattg gcattttaca gtattccaca gcgatgaatc aaaagcggag 180
cttcttcctg tagcgtataa tcaaaaacaa atcgttgagt cttctgctgt tgttgccatt 240
ttaggcgatt taaaggcaaa tgaaaacggt gaagaagttt atgctgaatt agcaagccaa 300
ggctatatta cggatgaaat caaacaaaca ttgctcggcc aaatcaacgg tgcttaccaa 360
agcgagcaat tcgcacgtga ttccgctttc ttaaatgctt ctttagctgc tatgcagctt 420
atgattgccg caaaagcaaa aggttatgac acttgcgcaa tcggcggatt taacaaagag 480
cagttccaaa agcaatttga tatcagtgag cgctatgttc cggttatgct tatttcaatc 540
ggcaaagcag tgaagcctgc gcatcaaagc aaccgtctgc cgctttcaaa agtatcaact 600
tggctgtaa 609
<210> 3
<211> 202
<212> PRT
<213> artificial sequence
<400> 3
Met Thr Asn Thr Leu Asp Val Leu Lys Ala Arg Ala Ser Val Lys Glu
1 5 10 15
Tyr Asp Thr Asp Ala Pro Ile Ser Lys Glu Glu Leu Thr Glu Leu Leu
20 25 30
Asp Leu Ala Thr Lys Ala Pro Ser Ala Trp Asn Leu Gln His Trp His
35 40 45
Phe Thr Val Phe His Ser Asp Glu Ser Lys Ala Glu Leu Leu Pro Val
50 55 60
Ala Asp Asn Gln Lys Gln Ile Val Glu Ser Ser Ala Val Val Ala Ile
65 70 75 80
Leu Gly Asp Leu Lys Ala Asn Glu Asn Gly Glu Glu Val Tyr Ala Glu
85 90 95
Leu Ala Ser Gln Gly Tyr Ile Thr Asp Glu Ile Lys Gln Thr Leu Leu
100 105 110
Gly Gln Ile Asn Gly Ala Tyr Gln Ser Glu Gln Phe Ala Arg Asp Ser
115 120 125
Ala Phe Leu Asn Ala Ser Leu Ala Ala Met Gln Leu Met Ile Ala Ala
130 135 140
Lys Ala Lys Gly Tyr Asp Thr Cys Ala Ile Gly Gly Phe Asn Lys Glu
145 150 155 160
Gln Phe Gln Lys Gln Phe Asp Ile Ser Glu Arg Tyr Val Pro Val Met
165 170 175
Leu Ile Ser Ile Gly Lys Ala Val Lys Pro Ala His Gln Ser Asn Arg
180 185 190
Leu Pro Leu Ser Lys Val Ser Thr Trp Leu
195 200
<210> 4
<211> 202
<212> PRT
<213> artificial sequence
<400> 4
Met Thr Asn Thr Leu Asp Val Leu Lys Ala Arg Ala Ser Val Lys Glu
1 5 10 15
Tyr Asp Thr Asp Ala Pro Ile Ser Lys Glu Glu Leu Thr Glu Leu Leu
20 25 30
Asp Leu Ala Thr Lys Ala Pro Ser Ala Trp Asn Leu Gln His Trp His
35 40 45
Phe Thr Val Phe His Ser Asp Glu Ser Lys Ala Glu Leu Leu Pro Val
50 55 60
Ala Tyr Asn Gln Lys Gln Ile Val Glu Ser Ser Ala Val Val Ala Ile
65 70 75 80
Leu Gly Asp Leu Lys Ala Asn Glu Asn Gly Glu Glu Val Tyr Ala Glu
85 90 95
Leu Ala Ser Gln Gly Tyr Ile Thr Asp Glu Ile Lys Gln Thr Leu Leu
100 105 110
Gly Gln Ile Glu Gly Ala Tyr Gln Ser Glu Gln Phe Ala Arg Asp Ser
115 120 125
Ala Phe Leu Asn Ala Ser Leu Ala Ala Met Gln Leu Met Ile Ala Ala
130 135 140
Lys Ala Lys Gly Tyr Asp Thr Cys Ala Ile Gly Gly Phe Asn Lys Glu
145 150 155 160
Gln Phe Gln Lys Gln Phe Asp Ile Ser Glu Arg Tyr Val Pro Val Met
165 170 175
Leu Ile Ser Ile Gly Lys Ala Val Lys Pro Ala His Gln Ser Asn Arg
180 185 190
Leu Pro Leu Ser Lys Val Ser Thr Trp Leu
195 200
Claims (10)
1. A mutant of NADH oxidase is characterized in that 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 mutating tyrosine at the 66 th position into aspartic acid;
or the asparagine at the 20 th position of NADH oxidase with the amino acid sequence shown in SEQ ID NO.1 is mutated into aspartic acid, and the asparagine at the 116 th position is mutated into glutamic acid.
2. A gene encoding the NADH oxidase mutant of claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. The recombinant vector according to claim 3, wherein the recombinant vector uses a pETDust series vector as a 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 according to claim 5, wherein the recombinant cell comprises E.coli as a host cell.
7. A method for reducing the optimal pH of NADH oxidase and improving the enzyme activity is characterized in that the method comprises the following steps of,
an asparagine at position 20 of NADH oxidase with an amino acid sequence shown in SEQ ID NO.1 is mutated into aspartic acid, and simultaneously tyrosine at position 66 is mutated into aspartic acid;
or the asparagine at the 20 th position of NADH oxidase with the amino acid sequence shown in SEQ ID NO.1 is mutated into aspartic acid, and the asparagine at the 116 th position is mutated into glutamic acid.
8. A method for regenerating coenzyme NAD+, characterized in that the mutant according to claim 1, or the recombinant cell according to claim 5 or 6 is added into a reaction system containing dissolved oxygen and NADH, and reacted to obtain NAD+.
9. The method according to claim 8, wherein the reaction conditions are: 20-28 ℃ and pH 7.0-7.5.
10. Use of the gene of claim 2, or the recombinant vector of claim 3 or 4, for increasing intracellular nad+ content of a microorganism, or for the preparation of a product that increases intracellular nad+ content of a microorganism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110908307.1A CN113667651B (en) | 2021-08-09 | 2021-08-09 | NADH oxidase mutant with improved enzyme activity and changed optimal pH |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110908307.1A CN113667651B (en) | 2021-08-09 | 2021-08-09 | NADH oxidase mutant with improved enzyme activity and changed optimal pH |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113667651A CN113667651A (en) | 2021-11-19 |
| CN113667651B true CN113667651B (en) | 2023-08-25 |
Family
ID=78541875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110908307.1A Active CN113667651B (en) | 2021-08-09 | 2021-08-09 | NADH oxidase mutant with improved enzyme activity and changed optimal pH |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113667651B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4221830A1 (en) * | 1991-07-25 | 1993-01-28 | Biotechnolog Forschung Gmbh | Escherichia coli expression vector for NADH-oxidase gene - derived from 26.8kD gene isolated from Thermus thermophilus, useful as highly stable bio-sensor |
| WO2009091054A1 (en) * | 2008-01-17 | 2009-07-23 | Keio University | Novel hydrogen peroxide-forming nadh oxidase, and dna encoding the same |
| WO2011090054A1 (en) * | 2010-01-20 | 2011-07-28 | 株式会社カネカ | Nadh oxidase mutant having improved stability and use thereof |
| KR20120078962A (en) * | 2011-01-03 | 2012-07-11 | 건국대학교 산학협력단 | A thermostable h2o forming nadh oxidase from lactobacillus rhamnosus and a preparation method thereof |
| KR101325057B1 (en) * | 2012-05-09 | 2013-11-06 | 건국대학교 산학협력단 | A nadh oxidase mutant improved in stability and activity |
-
2021
- 2021-08-09 CN CN202110908307.1A patent/CN113667651B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4221830A1 (en) * | 1991-07-25 | 1993-01-28 | Biotechnolog Forschung Gmbh | Escherichia coli expression vector for NADH-oxidase gene - derived from 26.8kD gene isolated from Thermus thermophilus, useful as highly stable bio-sensor |
| WO2009091054A1 (en) * | 2008-01-17 | 2009-07-23 | Keio University | Novel hydrogen peroxide-forming nadh oxidase, and dna encoding the same |
| JP2009165417A (en) * | 2008-01-17 | 2009-07-30 | Keio Gijuku | New hydrogen peroxide-forming type nadh oxidase and dna encoding the same |
| WO2011090054A1 (en) * | 2010-01-20 | 2011-07-28 | 株式会社カネカ | Nadh oxidase mutant having improved stability and use thereof |
| KR20120078962A (en) * | 2011-01-03 | 2012-07-11 | 건국대학교 산학협력단 | A thermostable h2o forming nadh oxidase from lactobacillus rhamnosus and a preparation method thereof |
| KR101325057B1 (en) * | 2012-05-09 | 2013-11-06 | 건국대학교 산학협력단 | A nadh oxidase mutant improved in stability and activity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113667651A (en) | 2021-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11332731B2 (en) | Nitrile hydratase mutant, genetically engineered bacterium containing mutant and applications thereof | |
| CN110938616B (en) | Mutant of nitrile hydratase derived from hot spring thermokalite bacillus | |
| US12104189B2 (en) | Mutant of nitrile hydratase derived from Caldalkalibacillus thermarum | |
| CN112877307B (en) | Amino acid dehydrogenase mutant and application thereof | |
| CN112391365B (en) | Starch branching enzyme mutant with improved catalytic activity and application thereof | |
| CN112322606B (en) | Nitrile hydratase mutant and application thereof | |
| CN113151237B (en) | Sucrose isomerase mutant with improved stability and construction method thereof | |
| CN112301012B (en) | Cyclodextrin glucosyltransferase mutant and construction method thereof | |
| CN110669751B (en) | Mutant zymoprotein of alanine racemase and preparation method thereof | |
| CN113667651B (en) | NADH oxidase mutant with improved enzyme activity and changed optimal pH | |
| CN116676296A (en) | Beta-1, 3-glucanase mutants with improved enzymatic activity | |
| CN114277022B (en) | Nitrile hydratase mutant with high activity and high thermal stability | |
| CN112226422B (en) | EstWY enzyme mutant with improved activity | |
| CN108624568B (en) | Mutant xanthine dehydrogenase and application thereof | |
| CN114621944B (en) | Arginine deiminase mutant with improved enzyme activity | |
| CN109370997B (en) | Phenylalanine aminomutase mutant | |
| CN114395543B (en) | Trehalose synthase mutant and application thereof | |
| CN112210544B (en) | Cyclodextrin glucosyltransferase mutant and application thereof | |
| CN109280651B (en) | Lactate dehydrogenase mutant gene LbLDH1 and fermentation method for efficient expression of lactate dehydrogenase mutant gene LbLDH1 in escherichia coli | |
| CN115786296B (en) | Meso-diaminopimelate dehydrogenase mutant and production method thereof | |
| CN109337891B (en) | Phenylalanine aminomutase mutant with improved thermal stability | |
| CN117165543A (en) | Alcohol dehydrogenase mutant derived from escherichia coli and application thereof | |
| CN116410948A (en) | Ethylene synthetase and application thereof | |
| CN117987402A (en) | L-aspartic acid alpha-decarboxylase mutants | |
| CN115948355A (en) | NADPH oxidase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |