CN115772514A - Modification of nitrile hydratase substrate channel amino acid motif for preparation of cinnamamide - Google Patents
Modification of nitrile hydratase substrate channel amino acid motif for preparation of cinnamamide Download PDFInfo
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Abstract
The invention discloses modification of a nitrile hydratase substrate channel amino acid motif for preparing cinnamamide, and belongs to the technical field of bioengineering. According to the invention, mutation is carried out on phenylalanine at the 37 th site on the nitrile hydratase beta subunit, leucine at the 48 th site and/or glutamine at the 89 th site on the alpha subunit, so that the enzyme activities of the mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N for catalyzing cinnamonitrile reach 46.5-53.6U/mL, and are improved by 9.3-10.72 times compared with the enzyme activity of wild nitrile hydratase. The mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N have good catalytic activity on cinnamonitrile and have good industrial application potential.
Description
Technical Field
The invention relates to modification of a nitrile hydratase substrate channel amino acid motif for preparing cinnamamide, and belongs to the technical field of bioengineering.
Background
The amide substance carrying the unique amide group has special physiological functions and potential biological activity, and is an advantageous resource for synthesizing a large amount of fine chemicals, polymers and synthetic intermediates. Currently, many synthetic methods have been developed for the preparation of amides. The synthesis of cinnamamides and their derivatives is the classical case among them and they can serve as carrier scaffolds for many natural products. For example, such scaffolds can be used as efficient templates for designing and producing novel pharmacologically active drug-like molecules. At present, the acquisition of cinnamamide and derivatives thereof is mainly based on traditional chemical synthesis in industrial production, and the literature on the catalysis of a biological enzyme method is very few. In the chemical production process, the operation is more complicated, and extreme temperature, strong base, toxic reagent triethylamine and the like are needed. The harsh reaction conditions have low safety factors and are inconsistent with the green production concept, so that improvement is urgently needed.
Nitriles are valuable intermediates in the synthesis, which can be relatively easily obtained from a number of simple, straightforward and cost-effective synthetic processes. Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme which can catalyze nitrile substances to be converted into amide compounds with high added values through hydration reaction, and is widely applied to the industrial production of nicotinamide and acrylamide. At present, the amide biological method production technology gradually replaces the traditional chemical method by the advantages of environmental protection, mild reaction conditions, high safety factor and the like, and accords with the sustainable development and green production concept.
Nitrile hydratases are generally composed of two subunits, namely alpha subunit and beta subunit, and researches show that most of the nitrile hydratases from prokaryotes reported at present have the problems of poor stability, low catalytic activity and narrow catalytic substrate spectrum. There have also been many studies attempting to modify them to improve their relevant properties. However, the problems of low catalytic efficiency, narrow substrate spectrum and the like at present still limit the further development and application of nitrile hydratase. Especially for substrates with larger steric hindrance like cinnamonitrile, the activity is lower.
Disclosure of Invention
Aiming at the technical difficulties and problems in the prior art, the invention modifies the substrate channel amino acid of nitrile hydratase derived from Pseudonocardia thermophila so as to broaden the substrate spectrum and improve the catalytic performance.
The invention provides a nitrile hydratase mutant, wherein the nitrile hydratase comprises an alpha subunit and a beta subunit, and nitrile hydratase PtNHase derived from pseudomonadacia thermophila is used as a parent; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2; the mutant is obtained by mutating at least one position of 37 phenylalanine of beta subunit, 48 leucine of beta subunit or 89 glutamine of alpha subunit.
In one embodiment, the mutant is any one of the following (a) to (e):
(a) The mutant is obtained by mutating 37 th phenylalanine and 48 th leucine of a beta subunit to be substituted;
(b) The mutant is obtained by replacing the 89 th glutamine mutation of alpha subunit;
(c) The mutant is obtained by mutating 37 th phenylalanine, 48 th leucine and 89 th glutamine of beta subunit;
(d) The mutant is obtained by mutating 37 th phenylalanine of a beta subunit and 89 th glutamine of an alpha subunit;
(e) The mutant is obtained by mutating leucine at position 48 of a beta subunit and glutamine at position 89 of an alpha subunit.
In one embodiment, the mutant is a beta subunit with a mutation at position 37 to proline or valine and a mutation at position 48 to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine, or threonine.
In one embodiment, the mutant is a beta subunit with a mutation at position 37 to proline and at position 48 to alanine proline or threonine.
In one embodiment, the mutant is a mutation of position 89 of the alpha subunit to cysteine, aspartic acid, lysine, asparagine, serine, valine.
In one embodiment, the mutant is a mutation of position 89 of the alpha subunit to asparagine.
The present invention provides a gene encoding the mutant.
The invention provides a recombinant plasmid carrying the gene.
In one embodiment, the recombinant plasmid is a pET series expression vector.
In one embodiment, the recombinant plasmid uses pET-24a (+) as an expression vector.
The invention provides host cells expressing the mutants, or containing the genes.
In one embodiment, the host cell comprises a prokaryotic or eukaryotic microorganism.
In one embodiment, the host cell is E.coli.
In one embodiment, the host cell is e.coli BL21 (DE 3).
The invention provides a recombinant strain expressing the nitrile hydratase mutant.
In one embodiment, the recombinant strain uses E.coli BL21 (DE 3) as a host and pET-24a (+) as an expression vector.
The invention provides a method for improving the catalytic activity of nitrile hydratase, which takes nitrile hydratase PtNHase derived from pseudomonadacia thermophila as a parent; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2; the mutant is obtained by mutating at least one of 37 th phenylalanine of beta subunit, 48 th leucine of beta subunit or 89 th glutamine of alpha subunit.
In one embodiment, the method is any one of the following (a) to (e):
(a) The method is characterized in that 37 th phenylalanine and 48 th leucine of a beta subunit are mutated and substituted;
(b) The method is to replace the 89 th glutamine mutation of alpha subunit;
(c) The method is to replace phenylalanine at position 37, leucine at position 48 and glutamine at position 89 of the beta subunit by mutation;
(d) The method is that phenylalanine at the 37 th position of the beta subunit and glutamine at the 89 th position of the alpha subunit are mutated to be substituted;
(e) The method is characterized in that the leucine at the 48 th position of the beta subunit and the glutamine at the 89 th position of the alpha subunit are mutated to be substituted.
In one embodiment, the method is a mutation of the beta subunit at position 37 to proline or valine and at position 48 to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine or threonine.
In one embodiment, the method is to mutate the beta subunit to proline at position 37 and alanine, proline or threonine at position 48.
In one embodiment, the method is mutation of position 89 of the alpha subunit to cysteine, aspartic acid, lysine, asparagine, serine, valine.
In one embodiment, the method is to mutate the alpha subunit to asparagine at position 89.
The invention provides a method for preparing cinnamamide, which takes cinnamonitrile as a substrate and utilizes the nitrile hydratase mutant to catalyze and generate cinnamamide.
The invention also provides application of the nitrile hydratase mutant, the gene, the recombinant plasmid or the host cell in preparation of cinnamamide.
In one embodiment, the gene on the expression vector is linked in the order of a gene encoding a β subunit gene, a gene encoding an α subunit gene, and a gene encoding a regulatory protein.
Has the beneficial effects that:
the invention finds that amino acid residues at positions 37 and 48 on a beta subunit and an amino acid residue at position 89 on an alpha subunit of Pt-NHase are possibly in a key structure domain of nitrile hydratase and have important effects on the catalytic activity of the nitrile hydratase, and provides a Pt-NHase mutant, wherein the mutant is obtained by mutating phenylalanine and leucine at positions 37 and 48 on the beta subunit relative to a wild type to obtain mutants F37P-L48A, F37P-L48P and F37P-L48T; the mutation of glutamine at position 89 on the alpha subunit resulted in mutant Q89N. For the catalysis of cinnamonitrile, the enzyme activities of the mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N are obviously improved compared with wild enzymes, are improved by more than 10 times to the maximum, further development and application of nitrile hydratase are developed, and great potential exists in industrial production of cinnamoamide.
Drawings
FIG. 1 shows the initial screening and enzyme activity determination of mutant library constructed by three amino acid residues of PtNHase substrate channel.
FIG. 2 shows the rescreening of PtNHase mutant library and the determination of enzyme activity.
Detailed Description
(I) culture Medium
LB culture medium: tryptone 10.0g/L, yeast extract 5.0g/L, naCl10.0g/L, kanamycin final concentration 50 u g/mL.
2 × YT medium: 16.0g/L of tryptone, 10.0g/L of yeast extract, 5.0g/L of NaCl5, and 50 mu g/mL of kanamycin final concentration.
(II) enzyme activity detection method
Enzyme activity (U) of nitrile hydratase: the unit enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1. Mu. Mol amide product from nitrile substrate per minute at 25 ℃.
Enzyme activity of nitrile hydratase (U/mL): the enzyme activity per ml of nitrile hydratase.
Example 1: construction of each mutant of Pt NHase
The gene coding the beta subunit (the amino acid sequence is shown as SEQ ID NO. 2), the gene coding the alpha subunit (the amino acid sequence is shown as SEQ ID NO. 1) and the gene coding the regulatory protein are sequentially connected between enzyme cutting sites NdeI (CATATG) and BamHI (GGATCC) of an expression vector pET-24a (+) in sequence, the genes are transformed into E.coli JM109, after LB plate culture, a single clone is selected and subjected to sequencing verification by Anthrada (Suzhou) Biotechnology Limited company to obtain a positive transformant, and a plasmid is extracted from the positive transformant, namely the wild plasmid pET24a (+) -PtNHase WT.
Firstly, using wild type plasmid pET24a (+) -PtNHase WT as a template, designing a mutation sequence on a primer, amplifying DNA fragments with mutation of a base sequence through PCR, wherein corresponding primer sequences used by different mutation plasmids are shown in Table 1, constructing by adopting a whole plasmid PCR method to obtain single-point mutant plasmids pET24a (+) -F37P, pET24a (+) -L48A, pET24a (+) -L48P, pET24a (+) -L48T and pET24a (+) -Q89N, and then respectively using the single-point mutation plasmids as the template to construct mutant plasmids pET24a (+) -F37P-L48A, pET24a (+) -F37P-L48P and pET24a (+) -F37P-L48T.
As shown in Table 2, the PCR amplification reaction conditions were pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min30s, and extension at 72 ℃ for 5min, for 30 cycles.
TABLE 1 primer sequences
TABLE 2 PCR amplification System
Example 2: catalytic efficiency of wild type and mutant of Pt NHase on cinnamonitrile
The wild-type plasmid p from example 1 was usedET24a (+) -PtNHaseWT and mutant plasmids pET24a (+) -F37P-L48A, pET24a (+) -F37P-L48P, pET24a (+) -F37P-L48T and pET24a (+) -Q89N are respectively transformed into E.coli BL21 (DE 3), a single colony is picked up to 5mLLB culture medium and cultured for 7-8h at 37 ℃ and 200 rpm. Transferring the seed solution to 100mL2 XYT medium at 1% (v/v), culturing at 37 deg.C and 200rpm to OD 600 To 0.6-0.8, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.4mM and CoCl at 0.1g/L 2 ·6H 2 And O, changing the culture temperature to 25 ℃, performing induced expression for 16h, and centrifuging to obtain wild type WT and mutant cells F37P-L48A, F37P-L48P, F37P-L48T and Q89N.
With 10mMKPB (by K) 2 HPO 4 :KH 2 PO 4 Formulation of =4, ph 7.4) buffer OD of WT and F37P-L48A, F37P-L48P, F37P-L48T and Q89N mutant cells 600 Adjusted to 0.5, 10. Mu.L to 1.5mL centrifuge tube was placed on a 25 ℃ metal bath. mu.L of substrate (5 mM cinnamonitrile solution containing 10% methanol) was added to the centrifuge tube, vortexed well, reacted at 25 ℃ for 5min, and then quenched by the addition of 500. Mu.L of pure acetonitrile. The reaction solution was passed through a 0.22 μm filter and subjected to liquid phase detection.
The liquid phase detection method comprises the following steps: the mobile phase composition is acetonitrile: water =1:2 (v/v), the flow rate is 1mL/min, the detection wavelength is 261nm, the column temperature is 40 ℃, and the generation amount of the product cinnamamide in the reaction system is determined.
The enzyme activity calculation results of WT and each mutant are shown in FIGS. 1 and 2, the enzyme activity of the wild enzyme WT is 5U/mL, and the enzyme activities of the mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N are 53.6U/mL, 50.1U/mL, 46.5U/mL and 47.0U/mL respectively.
That is, when the 37 th and 48 th amino acid residues on the beta subunit are mutated simultaneously or the 89 th amino acid residue on the alpha subunit is mutated, the enzyme activity of the nitrile hydratase is remarkably improved, which indicates that the 37 th, 48 th and 89 th amino acid residues may be in the key structure domain of the enzyme and have important function for the catalytic activity of the nitrile hydratase.
TABLE 3 comparison of the enzyme activities of the wild type and the mutant on cinnamonitrile
| Sample (I) | WT | F37P-L48A | F37P-L48P | F37P-L48T | Q89N |
| Enzyme activity (U/mL) | 5.0±1.0 | 53.6±2.7 | 50.1±3.0 | 46.5±2.5 | 47.0±2.1 |
Comparative example 1
The difference is that, for wild type PtNHase, single point saturation mutation is carried out, and phenylalanine at position 37 and leucine at position 48 on a beta subunit and glutamine at position 89 on an alpha subunit are respectively replaced by other 19 amino acids (mutation is respectively A/C/D/E/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y). After the mutant is cultured and induced to express, the cell is adjusted to the same OD (OD) of 0.5, the enzyme activity of the cell is measured, and the result shows that the enzyme activity is higher when the 37 th position is mutated into proline, the 48 th position is mutated into alanine, cysteine, histidine, lysine, proline, arginine and threonine, and the 89 th position is mutated into aspartic acid, asparagine, serine and valine (figure 1).
TABLE 4 comparison of Whole cell catalytic Activity of wild type and mutant on cinnamonitrile
| Sample (I) | Enzyme activity (U/mL) | Sample (I) | Enzyme activity (U/mL) | Sample (I) | Enzyme activity (U/mL) |
| WT | 5.0±1.0 | — | — | — | — |
| F37A | 5.0±1.0 | L48A | 28.1±2.5 | Q89A | 15.9±1.8 |
| F37C | 6.3±0.8 | L48C | 26.6±2.3 | Q89C | 25.5±2.0 |
| F37D | 6.4±1.0 | L48D | 13.9±1.9 | Q89D | 39.8±3.9 |
| F37E | 6.9±0.9 | L48E | 13.9±1.6 | Q89E | 29.9±3.0 |
| F37G | 9.8±1.2 | L48F | 19.7±2.1 | Q89F | 12.4±1.0 |
| F37H | 3.8±0.5 | L48G | 22.2±2.4 | Q89G | 20.4±2.0 |
| F37I | 10.0±1.0 | L48H | 25.6±2.1 | Q89H | — |
| F37K | 1.5±0.3 | L48I | 8.5±1.8 | Q89I | — |
| F37L | 8.5±1.2 | L48K | 27.9±2.6 | Q89K | 28.2±2.5 |
| F37M | 1.0±0.1 | L48M | — | Q89L | 29.4±3.0 |
| F37N | 8.2±1.5 | L48N | 21.2±1.4 | Q89M | 2.0±0.2 |
| F37P | 12.0±2.0 | L48P | 32.6±2.6 | Q89N | 47.0±4.5 |
| F37Q | 8.2±1.3 | L48Q | 19.7±2.0 | Q89P | — |
| F37R | 0.5±0.1 | L48R | 35.0±3.1 | Q89R | 20.7±1.9 |
| F37S | 6.3±1.1 | L48S | 11.2±2.1 | Q89S | 30.5±3.0 |
| F37T | 8.1±1.4 | L48T | 21.7±1.8 | Q89T | 14.4±1.4 |
| F37V | 10.6±1.7 | L48V | 21.8±1.9 | Q89V | 43.1±4.3 |
| F37W | 3.4±0.7 | L48W | 9.3±1.4 | Q89W | 10.7±1.1 |
| F37Y | 0.8±0.1 | L48Y | 17.4±2.0 | Q89Y | 1.5±0.2 |
Comparative example 2
See examples and comparative example 1, except that the second round of PCR was performed on the single-point mutants with higher activity in the above comparative example 1, and two mutants were obtained in combination. The results showed that the enzyme activities of F37P-L48A, F37P-L48H, F37P-L48P, F37P-L48T and Q89N-L48T were high (FIG. 2).
TABLE 5 comparison of the catalytic activity of the wild-type and mutant whole cells on cinnamonitrile
| Sample (I) | Enzyme activity (U/mL) | Sample (I) | Enzyme activity (U/mL) |
| WT | 5.0±1.0 | — | — |
| F37P-L48A | 55.7±4.0 | Q89D-L48A | 23.8±2.2 |
| F37P-L48C | 39.8±4.0 | Q89D-L48H | 13.4±1.3 |
| F37P-L48H | 36.9±4.2 | Q89D-L48P | 23.5±2.5 |
| F37P-L48K | 17.5±1.5 | Q89D-L48T | 23.8±2.4 |
| F37P-L48P | 46.5±2.9 | Q89N-L48A | 28.9±3.0 |
| F37P-L48R | 39.2±4.0 | Q89N-L48H | 5.1±0.9 |
| F37P-L48T | 47.2±4.5 | Q89N-L48P | 30.5±3.0 |
| F37P-Q89D | 25.9±2.3 | Q89N-L48T | 46.4±4.6 |
| F37P-Q89N | 29.8±3.0 | Q89S-L48A | 17.0±1.7 |
| F37P-Q89S | 29.5±3.0 | Q89S-L48H | 14.5±1.5 |
| F37P-Q89V | 26.0±2.7 | Q89S-L48P | 11.6±1.2 |
| Q89S-L48T | 22.3±2.3 | ||
| Q89V-L48A | 25.3±2.6 | ||
| Q89V-L48H | 3.8±0.5 | ||
| Q89V-L48P | 23.2±2.6 | ||
| Q89V-L48T | 23.9±2.4 |
Comparative example 3
Referring to the examples and comparative examples 1 and 2, the difference is that the combined mutants with higher activity in comparative example 2, namely F37P-L48A, F37P-L48P and F37P-L48T, are respectively taken as templates, and a third round of PCR is carried out, and the PCR is iterated with single-point mutants Q89D, Q89N, Q89S and Q89V, so as to obtain 12 three-point combined mutants. The results show that the enzyme activities of F37P-L48T-Q89S and F37P-L48T-Q89N are higher and are respectively 23.3U/mL and 15.9U/mL. However, compared with the 4 mutants in the example, the enzyme activities of F37P-L48A, F37P-L48P, F37P-L48T and Q89N are also significantly reduced (FIG. 2).
TABLE 6 comparison of the catalytic activity of whole cells of wild type and mutant on cinnamonitrile
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.
Claims (10)
1. A nitrile hydratase mutant comprising an α subunit and a β subunit, wherein the nitrile hydratase PtNHase derived from pseudomonandia thermophila is used as a parent; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown as SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 2; the mutant is obtained by mutating at least one of phenylalanine at position 37 of beta subunit, leucine at position 48 of beta subunit or glutamine at position 89 of alpha subunit.
2. The nitrile hydratase mutant according to claim 1, wherein the mutant is a beta subunit having a mutation at position 37 to proline or valine and a mutation at position 48 to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine or threonine; the mutant is obtained by mutating the 89 th position of the alpha subunit into cysteine, aspartic acid, lysine, asparagine, serine and valine.
3. A gene encoding the mutant of claim 1 or 2.
4. A recombinant plasmid carrying the gene of claim 3.
5. A host cell expressing the mutant of claim 1 or 2 or containing the gene of claim 3.
6. A recombinant strain expressing the nitrile hydratase mutant according to claim 1 or 2.
7. A method for improving the catalytic activity of nitrile hydratase is characterized in that nitrile hydratase PtNHase derived from Pseudonocardia thermophila is used as a parent; the amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown in SEQ ID NO. 2; the mutant is obtained by mutating at least one of 37 th phenylalanine of beta subunit, 48 th leucine of beta subunit or 89 th glutamine of alpha subunit.
8. The method of claim 7, wherein the beta subunit is mutated at position 37 to proline or valine, the 48 position to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine, or threonine, and the alpha subunit is mutated at position 89 to cysteine, aspartic acid, lysine, asparagine, serine, valine.
9. A method for producing cinnamamide, comprising catalyzing the production of cinnamamide using cinnamonitrile as a substrate using the mutant according to claim 1 or 2, or the recombinant strain according to claim 8.
10. Use of the mutant of claim 1 or 2, or the gene of claim 3, or the recombinant plasmid of claim 4, or the host cell of claim 5, or the recombinant strain of claim 6 for the preparation of cinnamamide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116240187A (en) * | 2023-04-06 | 2023-06-09 | 广州普言生物科技有限公司 | Prolyl hydroxylase alpha 1 subunit mutant, coding gene and application thereof in catalyzing hydroxylation of proline |
| CN116790573A (en) * | 2023-08-21 | 2023-09-22 | 清华大学 | Nitrile hydratase mutant and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116240187A (en) * | 2023-04-06 | 2023-06-09 | 广州普言生物科技有限公司 | Prolyl hydroxylase alpha 1 subunit mutant, coding gene and application thereof in catalyzing hydroxylation of proline |
| CN116240187B (en) * | 2023-04-06 | 2024-05-07 | 广东普言生物科技有限公司 | Prolyl hydroxylase alpha 1 subunit mutant, coding gene and application thereof in catalyzing hydroxylation of proline |
| CN116790573A (en) * | 2023-08-21 | 2023-09-22 | 清华大学 | Nitrile hydratase mutant and application thereof |
| CN116790573B (en) * | 2023-08-21 | 2023-11-21 | 清华大学 | Nitrile hydratase mutant and application thereof |
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