CN112322607B - Fusion type nitrile hydratase and application thereof - Google Patents
Fusion type nitrile hydratase and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion type nitrile hydratase and application thereof, belonging to the fields of gene engineering and enzyme engineering. The invention discloses a subunit fusion type nitrile hydratase with various improved stability, belonging to the field of genetic engineering. The invention takes nitrile hydratase derived from Pseudomonas putida NRRL 1886 as an initial object, and fuses beta subunits and alpha subunits of the nitrile hydratase by different types of linkers with different lengths to obtain various subunit fusion type nitrile hydratases, so that the stability is obviously improved. The method is simple and efficient, not only can the nitrile hydratase with remarkably improved stability be used for industrial production, but also the rule that gene fusion influences the stability of the nitrile hydratase is summarized, and theoretical research is carried out for improving the stability of the nitrile hydratase.
Description
Technical Field
The invention relates to a fusion type nitrile hydratase and application thereof, belonging to the fields of gene engineering and enzyme engineering.
Background
Nitrile hydratase (NHase) can catalyze nitrile compounds to synthesize corresponding amide products, and is widely applied to the industrial production of acrylamide and nicotinamide, in the industrial production, NHase is mainly applied to the production of high-purity nicotinamide and acrylamide, and the enzymatic production of acrylamide is a typical case of replacing a chemical synthesis method by a green biocatalysis method, and has many advantages such as: simple process, mild reaction condition, low energy consumption, less three wastes and the like, so that the Nhase is an important enzyme in industrial production.
However, currently, nhases have poor thermal stability in industrial application processes, for example, nhases derived from Pseudomonas chlororaphis B23, rhodococcus rhodochrous j1, rhodococcus sp.n-774 are only stable below 20 ℃, while the hydration process of nitriles is an exothermic reaction, so that in an industrial catalytic process, condensed water is required to reduce the reaction temperature, thereby causing energy waste; in addition, the instability of nitrile hydratase is also reflected in poor tolerance to substrates (nitrile organic compounds) and products (amide organic compounds), and nitrile hydratase is easily inactivated; for example, in the enzymatic preparation process of acrylamide, the nitrile hydratase has low tolerance to substrates and products, so that the final product acrylamide can only be maintained at a low level, which affects the subsequent acrylamide concentration process. With the continuous expansion of the market, the demand of amide compounds is increasing, so that how to improve the thermal stability and product tolerance of nitrile hydratase has become a focus of research.
In order to solve the problem of poor heat stability of NHase, various strategies are adopted by a plurality of subject groups, including optimization of catalytic process, screening and domestication of wild strains, molecular modification of NHase and the like; in the course of previous research, the inventor's group of subjects fused two subunits by covalent bond at gene level by molecular means, eliminating the possibility of subunit depolymerization, and although improving the stability of the fused nitrile hydratase, the half-life of the fused nitrile hydratase at 50 ℃ is only 26min at the maximum, which still cannot meet the industrial demand.
Disclosure of Invention
Aiming at the problems of poor tolerance to substrates and products and poor thermal stability in industrial application of nitrile hydratase, the invention adopts a gene synthesis strategy to fuse two subunits beta and alpha of NHase from Pseudomonas putida NRRL 1886 together through spiral or flexible or rigid connecting peptide to obtain the subunit fusion type NHase with greatly improved stability, and is expected to be applied to industrial production.
The invention firstly provides a fusion type nitrile hydratase, the fusion type nitrile hydratase simultaneously expresses an alpha subunit, a beta subunit and a regulation subunit, and the beta subunit and the alpha subunit of the fusion type nitrile hydratase are fused by a Linker; the general formula of the Linker is as follows: a (EAAAK) n A, or (GGSG) n Or (PA) n Wherein n is an integer of at least 1.
In one embodiment of the present invention, the fusion between the β -subunit and the α -subunit of the fusion-type nitrile hydratase is performed by Linker which links the C-terminus of the β -subunit and the N-terminus of the α -subunit.
In one embodiment of the invention, n in the formula of the Linker is as follows: n is more than or equal to 1 and less than or equal to 15.
In one embodiment of the invention, the nucleotide sequence of the Linker is selected from the sequences shown in SEQ ID NO. 5-22.
In one embodiment of the present invention, the amino acid sequence of the β subunit is shown in SEQ ID No.2, the amino acid sequence of the α subunit is shown in SEQ ID No.3, and the amino acid sequence of the regulatory subunit is shown in SEQ ID No. 4.
In one embodiment of the present invention, the nucleotide sequence of the β subunit is represented by SEQ ID No.23, the nucleotide sequence of the α subunit is represented by SEQ ID No.24, and the nucleotide sequence of the regulatory subunit is represented by SEQ ID No. 25.
The present invention also provides a gene encoding the above-described fusion-type nitrile hydratase.
The invention also provides a recombinant vector carrying the fusion type nitrile hydratase gene.
In one embodiment of the invention, the recombinant vector uses a pET-24a plasmid as an expression vector.
The invention also provides a recombinant cell expressing the fusion type nitrile hydratase.
In one embodiment of the present invention, the recombinant cell is an E.coli host cell.
The invention also provides a preparation method of the fusion type nitrile hydratase, which comprises the following steps: combining the above recombinant cellsInoculating to LB culture medium, culturing at 35-37 deg.C to OD 600 When the temperature is 0.6-0.8 ℃, adding an inducer IPTG to induce for 12-18h at 25 ℃, and expressing the fusion type nitrile hydratase.
In one embodiment of the present invention, the method is to inoculate the recombinant cells in LB expression medium containing kanamycin, and culture the cells at 37 ℃ and 200r/min with shaking until OD 600 When the concentration is 0.6-0.8, adding inducer IPTG to 0.1mM 2+ To 0.1mg/L, the fusion type nitrile hydratase was expressed by inducing at 25 ℃ for 12-18 hours.
In one embodiment of the present invention, the method further comprises collecting the bacterial cells of the recombinant cells, disrupting the bacterial cells, collecting the supernatant, membrane-filtering the supernatant, and separating the supernatant with a Strep Trap HP column to obtain a fused nitrile hydratase.
The invention also provides a method for improving the heat stability and/or tolerance of nitrile hydratase, which comprises the following steps: a β subunit and an α subunit fused to nitrile hydratase; the beta subunit and the alpha subunit of the fusion nitrile hydratase are connected through a Linker; the general formula of the Linker is as follows: a (EAAAK) n A, or (GGSG) n Or (PA) n Wherein n is an integer of at least 1.
In one embodiment of the invention, the value of n in the formula of the Linker is: n is more than or equal to 1 and less than or equal to 15.
In one embodiment of the invention, the nucleotide sequence of the Linker is shown in SEQ ID NO. 5-22.
In one embodiment of the present invention, the amino acid sequence of the β subunit is shown as SEQ ID No.2, the amino acid sequence of the α subunit is shown as SEQ ID No.3, and the amino acid sequence of the regulatory subunit is shown as SEQ ID No. 4.
The invention also provides a method for producing amide substances, which adds the fusion type nitrile hydratase into a reaction system containing nitrile organic matters for reaction.
In one embodiment of the invention, at least 0.4mg/mL of fusion-type nitrile hydratase is added to a nicotinonitrile solution with a final concentration of 200-400mmol/L to obtain a reaction system, and the reaction system is placed at 20-28 ℃ for reaction for 8-15min.
The invention also provides the application of the fusion type nitrile hydratase, the gene, the recombinant vector or the recombinant cell in catalyzing nitrile compounds to hydrate corresponding amide products.
Advantageous effects
(1) Compared with wild enzymes, the heat stability of the fusion nitrile hydratase is obviously improved, wherein the half-life of the fusion nitrile hydratase A8 is up to 139min, which is 7.7 times of that of the wild enzymes; the half-life of the fusion type nitrile hydratase B8 is up to 94min, which is 5.2 times of that of the wild enzyme; the half-life of the fusion-type nitrile hydratase C8 is up to 53min, which is nearly 3 times that of the wild enzyme. The fusion type nitrile hydratase with improved thermal stability provided by the invention can overcome the defect of low thermal stability of nitrile hydratase in industrial production, improve the yield and save the cost.
(2) By adopting the technical scheme provided by the invention, the substrate tolerance and the product tolerance are both obviously improved, wherein when the substrate 3-cyanopyridine is adopted for reaction, the relative enzyme activities of the fusion nitrile hydratase A8, B8 and C8 are improved to 84%, 89% and 86% from the lower relative enzyme activity (30%) when the wild enzyme is adopted for reaction; after the products of the nitrile hydratase A8, B8 and C8 are treated for 30min under the action of nicotinamide, the relative enzyme activity of the fusion nitrile hydratase A8, B8 and C8 is improved to 80%, 94% and 86% from the lower relative enzyme activity (77%) of the wild enzyme during the reaction.
Drawings
FIG. 1: SDS-PAGE of the wild-type fusion nitrile hydratase and A1, A2, A3, A4, A6, A8.
FIG. 2 is a schematic diagram: SDS-PAGE patterns of the wild-type fusion nitrile hydratase and B1, B2, B3, B4, B6, B8.
FIG. 3: SDS-PAGE patterns of the wild-type fusion nitrile hydratase and C1, C2, C3, C4, C6, C8.
FIG. 4: specific enzyme activities of wild-type nitrile hydratase and fusion-type nitrile hydratase.
FIG. 5: substrate tolerance for wild-type nitrile hydratase and fusion-type nitrile hydratase.
FIG. 6: product tolerance of wild-type nitrile hydratase and fusion-type nitrile hydratase.
Detailed Description
The enzymes A1 to A16 in the following examples are defined as: a fusion-type nitrile hydratase having a beta subunit and an alpha subunit linked by a helical Linker of the general formula A (EAAAK) n A; wherein A is alanine, E is glutamic acid, and K is lysine; n =1-12, and n is an integer.
The enzymes B1 to B16 in the following examples are defined as: a fusion-type nitrile hydratase having a beta subunit and an alpha subunit linked by a flexible Linker of the general formula (GGSG) n (ii) a Wherein G is glycine and S is serine; n =1-16, and n is an integer.
The enzymes C1 to C16 in the following examples are defined as: a fusion-type nitrile hydratase having a beta subunit and an alpha subunit linked by a rigid Linker of the general formula (PA) n (ii) a Wherein P is proline and A is alanine; n =1-16, and n is an integer.
The Linker involved in the following examples is summarized in Table 1.
Table 1: linker summary of fusion nitrile hydratase beta and alpha subunits
The media involved in the following examples are as follows:
LB liquid medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.
2YT medium: 16g/L of peptone, 10g/L of yeast extract and 5g/L of NaCl.
The detection methods referred to in the following examples are as follows:
and (3) detecting the enzyme activity of nitrile hydratase:
the reaction was carried out in a 1.5mL EP tube. Adding 10 mu L of nitrile hydratase with the concentration adjusted to 0.5mg/mL into 490 mu L of mixed solution containing 200mM nicotinonitrile and 10mM phosphate buffer solution to obtain a reaction system; the reaction system was left to react at 25 ℃ for 10min, and 500. Mu.L of acetonitrile was added to terminate the reaction. After the reaction product passes through a 0.22 μm membrane, the activity of the enzyme is measured by HPLC on a C18 column, the mobile phase is a mixed solution of acetonitrile and water (water: acetonitrile = 2), the detection wavelength is 220nm, the detection temperature is 40 ℃, the flow rate of the mobile phase is 0.6mL/min, the sample injection amount is 10 μ L, the detection time is 10min, and each group of experimental data is repeated at least 3 times.
Definition of enzyme activity (U): the amount of enzyme required to convert 3-cyanopyridine (nicotinonitrile) per minute to form 1. Mu. Mol/L of nicotinamide is defined as 1U.
Specific enzyme activity (U/mg): enzymatic activity per mg of NHase.
Definition of relative enzyme activity: the enzyme activity of the mutant enzyme was defined as 100% when it was reacted at pH =7.4 and a temperature of 30 ℃ for 10 minutes.
Nitrile hydratase reaction system: to 490. Mu.L of a 200mM substrate 3-cyanopyridine solution, 10. Mu.L of a pure enzyme solution at a concentration of 0.5mg/mL was added, and after reaction at 25 ℃ for 10min, the reaction was terminated with 500. Mu.L of acetonitrile, and passed through a 0.22. Mu.m membrane to prepare a sample for liquid phase measurement.
And (3) detection of the nitrile hydratase content:
the mobile phase was water, as detected by HPLC: acetonitrile = 1; the detection wavelength is 210nm, and the flow rate is 0.6ml/min; the chromatographic column is a C18 column.
Detection of nicotinonitrile consumption:
adding 10 mu L of pure enzyme solution with the final concentration of 0.5mg/mL into 250 mu L of nicotinamide solution with the concentration of 500mmol/L to obtain a reaction system; incubating the reaction system at 25 ℃ for 30min, adding 250 mu L of 40mM nicotinonitrile solution into the reaction system, reacting at 25 ℃ for 10min, and adding 500 mu L of acetonitrile to terminate the reaction to obtain a reaction product; after the reaction product passes through a 0.22 mu m film, determining the residual amount of the nicotinonitrile as a sample for liquid phase determination; wherein the initial concentration of the nicotinonitrile is 20mM, and the initial concentration of the nicotinonitrile minus the residual amount of the nicotinonitrile is the consumption of the nicotinonitrile.
Example 1: preparation of fused nitrile hydratase
(1) Construction of fusion-type nitrile hydratase:
chemically synthesizing a wild enzyme NHase gene WT with a nucleotide sequence shown as SEQ ID NO.1, cloning the gene at Nde I and EcoRI enzyme cutting sites of pET24a plasmid, and completing the process by Jinzhi Zhi Suzhou to obtain pET24a-Nhase-WT recombinant plasmid. Taking pET24a-Nhase-WT as a template, carrying out PCR reaction under the conditions shown in Table 3 by using primers shown in Table 2 to obtain a PCR product, transforming E.coli JM109 competent cells with the PCR product, carrying out Kinzseviru sequencing, and obtaining a plasmid with a correct sequencing result, namely a recombinant plasmid carrying a gene encoding the fusion type nitrile hydratase: pET24a-NHase-A1, pET24a-NHase-A2, pET24a-NHase-A3, pET24a-NHase-A4, pET24a-NHase-A6, pET24a-NHase-A8, pET24a-NHase-A12, pET24a-NHase-B1, pET24a-NHase-B2, pET24a-NHase-B3, pET24a-NHase-B4, pET24a-NHase-B6, pET24a-NHase-B8, pET24a-NHase-B16, pET24a-NHase-C1, pET24a-NHase-C2, pET24a-NHase-C3, pET24a-NHase-C4, pET24a-NHase-C6, pET24a-NHase-C8, NHase-C8; transforming the recombinant plasmid carrying the gene for encoding the fusion type nitrile hydratase and the recombinant plasmid pET24a-Nhase-WT carrying the wild enzyme into E.coli BL21 strain for expression, obtaining recombinant strains BL21/pET24a-Nhase-WT, BL21/pET24a-NHase-A1, BL21/pET24a-pET24a-NHase-A2, BL21/pET24a-pET24a-NHase-A3, BL21/pET24a-pET24a-NHase-A4, BL21/pET24a-pET24a-NHase-A6, BL21/pET24a-pET24a-NHase-A8, BL21/pET24a-pET24a-NHase-A12, BL21/pET24a-pET24a-NHase-B1, BL21/pET24a-pET24a-NHase-B2, BL21/pET24a-pET24a-NHase-B3 BL21/pET24a-pET24a-NHase-B4, BL21/pET24a-pET24a-NHase-B6, BL21/pET24a-pET24a-NHase-B8, BL21/pET24a-pET24a-NHase-B16, BL21/pET24a-pET24a-NHase-C1, BL21/pET24a-pET24a-NHase-C2, BL21/pET24a-pET24a-NHase-C3, BL21/pET24a-pET24a-NHase-C4, BL21/pET24a-pET24a-NHase-C6, BL21/pET24a-pET24a-NHase-C8, BL21/pET24a-pET24a-NHase-C16.
TABLE 2 primers
TABLE 3 Whole plasmid PCR amplification reaction System
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 2min; (30 cycles); 72. extend at deg.C for 10min. The PCR product was identified by agarose gel electrophoresis. Then, the PCR product is purified and digested and then transferred into escherichia coli BL21 competent cells.
(2) The recombinant Escherichia coli obtained in step (1) was inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured overnight with shaking at 37 ℃ and 200r/min, to obtain each seed solution.
Inoculating the seed solution into 100mL LB liquid medium containing 50 μ g/mL kanamycin at 1% (v/v), shake-culturing at 37 deg.C and 200r/min until OD600 is 0.6-0.8, adding inducer IPTG at final concentration of 0.1mM and Co at final concentration of 0.1mg/L 2+ Inducing the solution at 25 deg.C for 12-18h to obtain fermentation broth, centrifuging the obtained fermentation broth at 12000rpm, and discarding supernatant to obtain recombinant Escherichia coli.
(3) Respectively using the recombinant Escherichia coli thallus obtained in the step (2) with binding buffer solution (20 mmol/L Na) 2 HPO 4 280mmol/L NaCl and 6mmol/L KCl) is concentrated by 5 times, the cell disruption solution is obtained by ultrasonic disruption, the cell disruption solution is centrifugated for 40min under the condition of 12000rpm, the obtained supernatant is filtered by a 0.22 mu m filter membrane, and the crude enzyme solution is respectively obtained.
(4) Respectively purifying the crude enzyme liquid obtained in the step (3) by the following steps: a1 mL strep Trap HP column was equilibrated with 10 column volumes of binding buffer, non-specifically adsorbed proteins were washed with 15 column volumes of binding buffer, and 20mM Na was used in 8 column volumes 2 HPO 4 280mM NaCl,6mM KCl,2.5mM desthiobiotin buffer solution elution proteins, and respectively collecting samples, namely purified wild-type nitrile hydratase WT, fusion nitrile hydratase A1, A2, A3, A4, A6, A8, A12, B1, B2, B3, B4, B6, B8, B16, C1, C2, C3, C46. C8 and C16, and identified by SDS-PAGE analysis (wherein SDS-PAGE of purified wild-type nitrile hydratase WT and fusion-type nitrile hydratase A1, A2, A3, A4, A6, A8, B1, B2, B3, B4, B6, B8, C1, C2, C3, C4, C6, C8 is shown in FIGS. 1 to 3), and as a result: protein bands were found at 49.2kDa, demonstrating that nitrile hydratase protein was expressed.
Example 2: detection of fusion-type nitrile hydratase enzyme activity
The enzyme activities of the purified wild-type nitrile hydratase WT, the fusion-type nitrile hydratases A1, A2, A3, A4, A6, A8, A12, B1, B2, B3, B4, B6, B8, B16, C1, C2, C3, C4, C6, C8, and C16 obtained in example 1 were measured, respectively, and the results are shown in Table 4;
table 4: enzyme activities of different nitrile hydratases
| Nitrile hydratase | Enzyme activity (U/mg) |
| WT | 488.32 |
| A1 | 627.25 |
| A2 | 526.83 |
| A3 | 638.50 |
| A4 | 653.76 |
| A6 | 691.98 |
| A8 | 355.36 |
| A12 | 345.31 |
| B1 | 489.60 |
| B2 | 536.97 |
| B3 | 511.20 |
| B4 | 607.42 |
| B6 | 524.24 |
| B8 | 599.25 |
| B16 | 447.67 |
| C1 | 630.57 |
| C2 | 546.24 |
| C3 | 603.07 |
| C4 | 586.10 |
| C6 | 551.49 |
| C8 | 540.26 |
| C16 | 443.89 |
As shown in table 4 and fig. 4, the enzyme activity of the fusion-type nitrile hydratase A6 was 691.98, which was increased by 42% as compared with the wild enzyme, the enzyme activity of the fusion-type nitrile hydratase B4 was 607.42, which was increased by 24% as compared with the wild enzyme, the enzyme activity of the fusion-type nitrile hydratase C1 was 630.57, and the enzyme activity was increased by 29% as compared with the wild enzyme.
Furthermore, as is clear from Table 4, the enzyme activities of the fusion-type nitrile hydratases A8 and A12 were lower than that of the wild-type enzyme, and the fusion-type nitrile hydratases B16 and C16 were both lower than that of the wild-type enzyme; therefore, the Linker length can improve the activity of the nitrile hydratase enzyme to different degrees within a certain range, but the activity of the enzyme can be reduced if the Linker is too long.
Example 3: detection of thermal stability of fusion-type nitrile hydratase
The heat stability of the fusion-type nitrile hydratases of the invention is improved, and the heat stability is measured by taking the fusion-type nitrile hydratases A1, A4, A8, B1, B4, B8, C1, C4 and C8 as examples, and the steps are as follows:
10. Mu.L of the wild-type nitrile hydratase WT and the fusion-type nitrile hydratase A1, A4, A8, B1, B4, B8, C1, C4, C8 purified in example 1 at a final concentration of 0.5mg/mL were treated in a metal bath at 50 ℃ for 15, 30, 45, 60, 75, 90min, respectively, and then the relative enzyme activities of the respective nitrile hydratases were determined, the enzyme activities of the respective enzymes were defined as 100% when not treated in the metal bath, and the half-lives t and t were calculated 1/2 (min), the results are shown in Table 5, and the thermal stability is analyzed.
TABLE 5 half-lives of the different nitrile hydratases
As is clear from Table 5, the half-life of the enzyme A8 was 139min, which was 7.7 times as long as that of the wild-type enzyme WT, the half-life of the enzyme B8 was 94min, which was 7.7 times as long as that of the wild-type enzyme WT, and the half-life of the enzyme C8 was 53min, which was 2.9 times as long as that of the wild-type enzyme WT. Therefore, different types of linkers have the advantages that the longer the Linker is, the better the heat stability of nitrile hydratase is, and the better the stability improvement effect of the spiral Linker is.
Example 4: detection of substrate tolerance of fused nitrile hydratase
The substrate tolerance of the fusion type nitrile hydratase provided by the invention is improved, and the fusion type nitrile hydratase A8, B8 and C8 are taken as examples, and the specific steps are as follows:
(1) To 490. Mu.L of a 200mmol/L nicotinonitrile-containing solution, 10. Mu.L of the purified wild-type nitrile hydratase WT, fusion-type nitrile hydratase A8, B8, and C8 obtained in example 1 was added to the solution to a final concentration of 0.5mg/mL to obtain a type I reaction system;
(2) To 490. Mu.L of a 500mmol/L nicotinonitrile-containing solution, 10. Mu.L of the purified wild-type nitrile hydratase WT, fusion-type nitrile hydratase A8, B8, and C8 obtained in example 1 was added to the solution to a final concentration of 0.5mg/mL to obtain a type II reaction system;
(3) After each reaction system obtained in the steps (1) and (2) is respectively placed at 25 ℃ for reaction for 10min, 500 mu L of acetonitrile is respectively added for stopping the reaction, the enzyme activity is measured and the substrate tolerance is reacted, the enzyme activity of 200mM substrate reaction in the reaction system 1 is defined as 100%, the enzyme activity of 500mM substrate reaction in the reaction system 2 is measured and the relative enzyme activity is calculated, and the results are shown in Table 6 and figure 5.
Table 6: relative enzyme activities of different nitrile hydratases
As is clear from Table 6 and FIG. 5, it was found that the relative enzyme activities of the fused nitrile hydratases A8, B8, and C8 were improved from 30% to 89%, 90%, and 86% of the wild enzymes by reacting the fused nitrile hydratase with 500mM of the substrate 3-cyanopyridine (nicotinonitrile), and it was found that the substrate nicotinonitrile tolerance of the fused nitrile hydratase was significantly improved.
Example 5: detection of product tolerance of fused nitrile hydratase
The substrate tolerance of the fusion type nitrile hydratase provided by the invention is improved, and the fusion type nitrile hydratase A8, B8 and C8 are taken as examples, and the specific steps are as follows:
to a solution containing 250. Mu.L of nicotinamide at a concentration of 500mmol/L, 10. Mu.L of the purified wild-type nitrile hydratase WT and the fusion-type nitrile hydratase fusion-type nitrile hydratases A8, B8, and C8 obtained in example 1 were added to each other to give a reaction system; incubating each reaction system at 25 ℃ for 30min, adding 250 mu L of 40mM nicotinonitrile solution into the reaction system, reacting at 25 ℃ for 10min, adding 500 mu L of acetonitrile to terminate the reaction, and reacting the catalytic capacity of the nicotinonitrile by measuring the consumption of the nicotinonitrile to obtain a product tolerance result; the consumption of nicotinonitrile at 0M treatment was defined as 100%, and the relative consumption of nicotinonitrile after 500mmol/L nicotinamide solution treatment, i.e., relative enzyme activity, was calculated, and the results are shown in Table 7 and FIG. 6.
Table 7: relative consumption of nicotinonitrile by different nitrile hydratases
| Sample name | Relative enzyme activity |
| WT | 77 |
| A8 | |
| 80％ | |
| B8 | 94％ |
| C8 | 86％ |
As is clear from Table 7 and FIG. 6, the relative enzyme activities of the fusion-type nitrile hydratases A8, B8, and C8 were improved from 77% of the wild enzymes to 80%, 94%, and 86% after 30min treatment with 500mM of the product nicotinamide, and the product tolerance of the fusion-type nitrile hydratases was significantly improved.
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
<110> university in south of the Yangtze river
<120> fusion type nitrile hydratase and application thereof
<130> BAA201071A
<160> 25
<170> PatentIn version 3.3
<210> 1
<211> 1801
<212> DNA
<213> Artificial sequence
<400> 1
atggcaagct ggagccaccc gcagttcgaa aagggtgcac atatgaatgg cattcacgat 60
actggcggag cacatggtta tgggccggtt tacagagaac cgaacgaacc cgtctttcgc 120
tacgactggg aaaaaacggt catgtccctg ctcccggcgc tgctcgccaa cggcaacttc 180
aacctcgatg aatttcggca ttcgatcgag cgaatgggcc cggcccacta tctggaggga 240
acctactacg aacactggct tcatgtcttt gagaacctgc tggtcgagaa gggtgtgctc 300
acggccacgg aagtcgcgac cggcaaggct gcgtctggca agacggcgac gccggtgctg 360
acgccggcca tcgtggacgg actgctcagc accggggctt ctgccgcccg ggaggagggt 420
gcgcgggcgc ggttcgctgt gggggacaag gttcgcgtcc tcaacaagaa cccggtgggc 480
catacccgca tgccgcgcta cacgcggggc aaagtgggga cagtggtcat cgaccatggt 540
gtgttcgtga cgccggacac cgcggcacac ggaaagggcg agcaccccca gcacgtttac 600
accgtgagtt tcacgtcggt cgaactgtgg gggcaagacg cttcctcgcc gaaggacacg 660
attcgcgtcg acttgtggga tgactacctg gagccagcgt gaaaggagac cgcaccatgg 720
ggcaatcaca cacgcatgac caccatcacg acgggtacca ggcaccgccc gaagacatcg 780
cgctgcgggt caaggccttg gagtctctgc tgatcgagaa aggtcttgtc gacccagcgg 840
ccatggactt ggtcgtccaa acgtatgaac acaaggtagg cccccgaaac ggcgccaaag 900
tcgtggccaa ggcctgggtg gaccctgcct acaaggcccg tctgctggca gacggcactg 960
ccggcattgc cgagctgggc ttctccgggg tacagggcga ggacatggtc attctggaaa 1020
acacccccgc cgtccacaac gtcttcgttt gcaccttgtg ctcttgctac ccatggccga 1080
cgctgggctt gccccctgcc tggtacaagg ccgcgcccta ccggtcccgc atggtgagcg 1140
acccgcgtgg ggttctcgcg gagttcggcc tggtgatccc cgccaacaag gaaatccgcg 1200
tctgggacac cacggccgaa ttgcgctaca tggtgctgcc ggaacggccc gcgggaactg 1260
aagcctacag cgaagaacaa ctggccgaac tcgttacccg cgattcgatg atcggcaccg 1320
gcctgcccac ccaacccacc ccatctcatt aaaaggagat atagatatga aagacgaacg 1380
gtttccattg ccagagggtt cgctgaagga cctcgatggc cctgtgtttg acgagccttg 1440
gcagtcccag gcgtttgcct tggtggtcag catgcacaag gccggtctct ttcagtggaa 1500
agactgggcc gagaccttca ccgccgaaat cgacgcttcc ccggctctgc ccggcgaaag 1560
cgtcaacgac acctactacc ggcaatgggt gtcggcgctg gaaaagttgg tggcgtcgct 1620
ggggcttgtg acgggtggag acgtcaactc gcgcgcacag gagtggaaac aggcccacct 1680
caacacccca catgggcacc cgatcctgct ggcccatgcg ctttgcccgc cagcgatcga 1740
ccccaagcac aagcacgagc cacaacgctc accgatcaag gtcgttgccg caatggcttg 1800
a 1801
<210> 2
<211> 233
<212> PRT
<213> Artificial sequence
<400> 2
Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala His Met Asn
1 5 10 15
Gly Ile His Asp Thr Gly Gly Ala His Gly Tyr Gly Pro Val Tyr Arg
20 25 30
Glu Pro Asn Glu Pro Val Phe Arg Tyr Asp Trp Glu Lys Thr Val Met
35 40 45
Ser Leu Leu Pro Ala Leu Leu Ala Asn Gly Asn Phe Asn Leu Asp Glu
50 55 60
Phe Arg His Ser Ile Glu Arg Met Gly Pro Ala His Tyr Leu Glu Gly
65 70 75 80
Thr Tyr Tyr Glu His Trp Leu His Val Phe Glu Asn Leu Leu Val Glu
85 90 95
Lys Gly Val Leu Thr Ala Thr Glu Val Ala Thr Gly Lys Ala Ala Ser
100 105 110
Gly Lys Thr Ala Thr Pro Val Leu Thr Pro Ala Ile Val Asp Gly Leu
115 120 125
Leu Ser Thr Gly Ala Ser Ala Ala Arg Glu Glu Gly Ala Arg Ala Arg
130 135 140
Phe Ala Val Gly Asp Lys Val Arg Val Leu Asn Lys Asn Pro Val Gly
145 150 155 160
His Thr Arg Met Pro Arg Tyr Thr Arg Gly Lys Val Gly Thr Val Val
165 170 175
Ile Asp His Gly Val Phe Val Thr Pro Asp Thr Ala Ala His Gly Lys
180 185 190
Gly Glu His Pro Gln His Val Tyr Thr Val Ser Phe Thr Ser Val Glu
195 200 205
Leu Trp Gly Gln Asp Ala Ser Ser Pro Lys Asp Thr Ile Arg Val Asp
210 215 220
Leu Trp Asp Asp Tyr Leu Glu Pro Ala
225 230
<210> 3
<211> 211
<212> PRT
<213> Artificial sequence
<400> 3
Met Gly Gln Ser His Thr His Asp His His His Asp Gly Tyr Gln Ala
1 5 10 15
Pro Pro Glu Asp Ile Ala Leu Arg Val Lys Ala Leu Glu Ser Leu Leu
20 25 30
Ile Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Leu Val Val Gln
35 40 45
Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala
50 55 60
Lys Ala Trp Val Asp Pro Ala Tyr Lys Ala Arg Leu Leu Ala Asp Gly
65 70 75 80
Thr Ala Gly Ile Ala Glu Leu Gly Phe Ser Gly Val Gln Gly Glu Asp
85 90 95
Met Val Ile Leu Glu Asn Thr Pro Ala Val His Asn Val Phe Val Cys
100 105 110
Thr Leu Cys Ser Cys Tyr Pro Trp Pro Thr Leu Gly Leu Pro Pro Ala
115 120 125
Trp Tyr Lys Ala Ala Pro Tyr Arg Ser Arg Met Val Ser Asp Pro Arg
130 135 140
Gly Val Leu Ala Glu Phe Gly Leu Val Ile Pro Ala Asn Lys Glu Ile
145 150 155 160
Arg Val Trp Asp Thr Thr Ala Glu Leu Arg Tyr Met Val Leu Pro Glu
165 170 175
Arg Pro Ala Gly Thr Glu Ala Tyr Ser Glu Glu Gln Leu Ala Glu Leu
180 185 190
Val Thr Arg Asp Ser Met Ile Gly Thr Gly Leu Pro Thr Gln Pro Thr
195 200 205
Pro Ser His
210
<210> 4
<211> 144
<212> PRT
<213> Artificial sequence
<400> 4
Met Lys Asp Glu Arg Phe Pro Leu Pro Glu Gly Ser Leu Lys Asp Leu
1 5 10 15
Asp Gly Pro Val Phe Asp Glu Pro Trp Gln Ser Gln Ala Phe Ala Leu
20 25 30
Val Val Ser Met His Lys Ala Gly Leu Phe Gln Trp Lys Asp Trp Ala
35 40 45
Glu Thr Phe Thr Ala Glu Ile Asp Ala Ser Pro Ala Leu Pro Gly Glu
50 55 60
Ser Val Asn Asp Thr Tyr Tyr Arg Gln Trp Val Ser Ala Leu Glu Lys
65 70 75 80
Leu Val Ala Ser Leu Gly Leu Val Thr Gly Gly Asp Val Asn Ser Arg
85 90 95
Ala Gln Glu Trp Lys Gln Ala His Leu Asn Thr Pro His Gly His Pro
100 105 110
Ile Leu Leu Ala His Ala Leu Cys Pro Pro Ala Ile Asp Pro Lys His
115 120 125
Lys His Glu Pro Gln Arg Ser Pro Ile Lys Val Val Ala Ala Met Ala
130 135 140
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<400> 5
tgcttctgct gctgcttttg c 21
<210> 6
<211> 36
<212> DNA
<213> Artificial sequence
<400> 6
tgcttctgct gctgcttttt ctgctgctgc ttttgc 36
<210> 7
<211> 51
<212> DNA
<213> Artificial sequence
<400> 7
tgcttctgct gctgcttttt ctgctgctgc tttttctgct gctgcttttg c 51
<210> 8
<211> 66
<212> DNA
<213> Artificial sequence
<400> 8
tgcttctgct gctgcttttt ctgctgctgc tttttctgct gctgcttttt ctgctgctgc 60
ttttgc 66
<210> 9
<211> 96
<212> DNA
<213> Artificial sequence
<400> 9
tgcttctgct gctgcttttt ctgctgctgc tttttctgct gctgcttttt ctgctgctgc 60
tttttctgct gctgcttttt ctgctgctgc ttttgc 96
<210> 10
<211> 126
<212> DNA
<213> Artificial sequence
<400> 10
tgcttctgct gctgcttttt ctgctgctgc tttttctgct gctgcttttt ctgctgctgc 60
tttttctgct gctgcttttt ctgctgctgc tttttctgct gctgcttttt ctgctgctgc 120
ttttgc 126
<210> 11
<211> 12
<212> DNA
<213> Artificial sequence
<400> 11
accgctacca cc 12
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence
<400> 12
accgctacca ccaccgctac cacc 24
<210> 13
<211> 36
<212> DNA
<213> Artificial sequence
<400> 13
accgctacca ccaccgctac caccaccgct accacc 36
<210> 14
<211> 48
<212> DNA
<213> Artificial sequence
<400> 14
accgctacca ccaccgctac caccaccgct accaccaccg ctaccacc 48
<210> 15
<211> 72
<212> DNA
<213> Artificial sequence
<400> 15
accgctacca ccaccgctac caccaccgct accaccaccg ctaccaccac cgctaccacc 60
accgctacca cc 72
<210> 16
<211> 96
<212> DNA
<213> Artificial sequence
<400> 16
accgctacca ccaccgctac caccaccgct accaccaccg ctaccaccac cgctaccacc 60
accgctacca ccaccgctac caccaccgct accacc 96
<210> 17
<211> 6
<212> DNA
<213> Artificial sequence
<400> 17
tgccgg 6
<210> 18
<211> 12
<212> DNA
<213> Artificial sequence
<400> 18
tgccggtgcc gg 12
<210> 19
<211> 18
<212> DNA
<213> Artificial sequence
<400> 19
<210> 20
<211> 24
<212> DNA
<213> Artificial sequence
<400> 20
tgccggtgcc ggtgccggtg ccgg 24
<210> 21
<211> 36
<212> DNA
<213> Artificial sequence
<400> 21
tgccggtgcc ggtgccggtg ccggtgccgg tgccgg 36
<210> 22
<211> 48
<212> DNA
<213> Artificial sequence
<400> 22
tgccggtgcc ggtgccggtg ccggtgccgg tgccggtgcc ggtgccgg 48
<210> 23
<211> 702
<212> DNA
<213> Artificial sequence
<400> 23
atggcaagct ggagccaccc gcagttcgaa aagggtgcac atatgaatgg cattcacgat 60
actggcggag cacatggtta tgggccggtt tacagagaac cgaacgaacc cgtctttcgc 120
tacgactggg aaaaaacggt catgtccctg ctcccggcgc tgctcgccaa cggcaacttc 180
aacctcgatg aatttcggca ttcgatcgag cgaatgggcc cggcccacta tctggaggga 240
acctactacg aacactggct tcatgtcttt gagaacctgc tggtcgagaa gggtgtgctc 300
acggccacgg aagtcgcgac cggcaaggct gcgtctggca agacggcgac gccggtgctg 360
acgccggcca tcgtggacgg actgctcagc accggggctt ctgccgcccg ggaggagggt 420
gcgcgggcgc ggttcgctgt gggggacaag gttcgcgtcc tcaacaagaa cccggtgggc 480
catacccgca tgccgcgcta cacgcggggc aaagtgggga cagtggtcat cgaccatggt 540
gtgttcgtga cgccggacac cgcggcacac ggaaagggcg agcaccccca gcacgtttac 600
accgtgagtt tcacgtcggt cgaactgtgg gggcaagacg cttcctcgcc gaaggacacg 660
attcgcgtcg acttgtggga tgactacctg gagccagcgt ga 702
<210> 24
<211> 636
<212> DNA
<213> Artificial sequence
<400> 24
atggggcaat cacacacgca tgaccaccat cacgacgggt accaggcacc gcccgaagac 60
atcgcgctgc gggtcaaggc cttggagtct ctgctgatcg agaaaggtct tgtcgaccca 120
gcggccatgg acttggtcgt ccaaacgtat gaacacaagg taggcccccg aaacggcgcc 180
aaagtcgtgg ccaaggcctg ggtggaccct gcctacaagg cccgtctgct ggcagacggc 240
actgccggca ttgccgagct gggcttctcc ggggtacagg gcgaggacat ggtcattctg 300
gaaaacaccc ccgccgtcca caacgtcttc gtttgcacct tgtgctcttg ctacccatgg 360
ccgacgctgg gcttgccccc tgcctggtac aaggccgcgc cctaccggtc ccgcatggtg 420
agcgacccgc gtggggttct cgcggagttc ggcctggtga tccccgccaa caaggaaatc 480
cgcgtctggg acaccacggc cgaattgcgc tacatggtgc tgccggaacg gcccgcggga 540
actgaagcct acagcgaaga acaactggcc gaactcgtta cccgcgattc gatgatcggc 600
accggcctgc ccacccaacc caccccatct cattaa 636
<210> 25
<211> 435
<212> DNA
<213> Artificial sequence
<400> 25
atgaaagacg aacggtttcc attgccagag ggttcgctga aggacctcga tggccctgtg 60
tttgacgagc cttggcagtc ccaggcgttt gccttggtgg tcagcatgca caaggccggt 120
ctctttcagt ggaaagactg ggccgagacc ttcaccgccg aaatcgacgc ttccccggct 180
ctgcccggcg aaagcgtcaa cgacacctac taccggcaat gggtgtcggc gctggaaaag 240
ttggtggcgt cgctggggct tgtgacgggt ggagacgtca actcgcgcgc acaggagtgg 300
aaacaggccc acctcaacac cccacatggg cacccgatcc tgctggccca tgcgctttgc 360
ccgccagcga tcgaccccaa gcacaagcac gagccacaac gctcaccgat caaggtcgtt 420
gccgcaatgg cttga 435
Claims (8)
1. A fusion type nitrile hydratase is characterized in that the fusion type nitrile hydratase simultaneously expresses an alpha subunit, a beta subunit and a regulation subunit, and the beta subunit and the alpha subunit of the fusion type nitrile hydratase are fused by a Linker; the general formula of the Linker is as follows: a (EAAAK) n A, or (GGSG) n Or (PA) n Wherein n is 8; the amino acid sequence of the beta subunit is shown as SEQ ID NO.2, the amino acid sequence of the alpha subunit is shown as SEQ ID NO.3, and the amino acid sequence of the regulatory subunit is shown as SEQ ID NO. 4; the fusion is linking the C-terminus of the β subunit and the N-terminus of the α subunit.
2. A gene encoding the fusion-type nitrile hydratase according to claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. A recombinant cell expressing the fused nitrile hydratase of claim 1.
5. The recombinant cell of claim 4, wherein the recombinant cell is an E.coli host cell.
6. A method for increasing the thermostability and/or tolerance of nitrile hydratase, the method comprising: a β subunit and an α subunit fused to nitrile hydratase; the beta subunit and the alpha subunit of the fusion nitrile hydratase are connected through a Linker; the general formula of the Linker is as follows: a (EAAAK) n A, or (GGSG) n Or (PA) n Wherein n is 8; the amino acid sequence of the beta subunit is shown as SEQ ID NO.2, the amino acid sequence of the alpha subunit is shown as SEQ ID NO.3, and the amino acid sequence of the regulatory subunit is shown as SEQ ID NO. 4; the fusion is linking the C-terminus of the β subunit and the N-terminus of the α subunit.
7. A method for producing an amide-based substance, characterized in that the fusion-type nitrile hydratase according to claim 1 is added to a reaction system containing a nitrile-based organic substance to effect a reaction.
8. Use of the fused nitrile hydratase according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4 or 5 for catalyzing the hydration of nitrile compounds to the corresponding amides.
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| CN102517271A (en) * | 2011-12-13 | 2012-06-27 | 清华大学 | Mutant nitrile hydratase |
| CN104450657A (en) * | 2014-11-06 | 2015-03-25 | 浙江大学 | Nitrile hydratase as well as encoding gene and application thereof |
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| AU2015270032B2 (en) * | 2014-06-06 | 2018-11-29 | Mitsubishi Chemical Corporation | Improved nitrile hydratase |
| CN104862296B (en) * | 2015-04-22 | 2017-12-12 | 江南大学 | A kind of specific enzyme activity and stability-enhanced pattern of fusion nitrile hydratase |
| CA3029627A1 (en) * | 2016-07-01 | 2018-01-04 | Resolve Therapeutics, Llc | Optimized binuclease fusions and methods |
| CN106986922B (en) * | 2017-04-14 | 2020-03-06 | 江南大学 | Self-assembled amphiphilic short peptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102517271A (en) * | 2011-12-13 | 2012-06-27 | 清华大学 | Mutant nitrile hydratase |
| CN104450657A (en) * | 2014-11-06 | 2015-03-25 | 浙江大学 | Nitrile hydratase as well as encoding gene and application thereof |
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