CN111690675A - Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof - Google Patents
Recombinant bacterium for expressing nitrile hydratase mutant and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant bacterium for expressing nitrile hydratase mutant and a preparation method and application thereof. The invention uses the genome of Escherichia coli with high nitrile hydratase yield as a template, adopts error-prone PCR technology to recombine and construct a random mutant plasmid library, screens nitrile hydratase mutant genes from the library, and can efficiently induce and express nitrile hydratase mutant proteins after the recombinant genes are transformed into engineering bacteria. The recombinant engineering bacteria of the high expression nitrile hydratase mutant coding gene are induced and expressed to obtain thallus which is used as an enzyme source, hydration reaction is carried out under a proper condition to produce nicotinamide, and the byproduct nicotinic acid is over 90 percent lower.
Description
Technical Field
The invention relates to the technical field of microbial gene recombination, in particular to a recombinant bacterium for expressing a nitrile hydratase mutant and a preparation method and application thereof.
Background
Directed evolution belongs to irrational design, and refers to the technical scheme that a natural evolution process of Darwinian is simulated in a laboratory, a coding gene of a certain protease is modified by error-prone PCR, DNA recombination and other technologies, and then valuable mutant enzymes are screened according to specific modification purposes. In the last 10 years, directed evolution technology has been successful in the field of modification of relevant properties of enzymes, mainly focusing on improving catalytic activity, improving substrate specificity, improving thermal stability, and the like.
At present, the production of nicotinamide is commonly carried out by biocatalytic production by recombinant engineering bacteria for expressing nitrile hydratase protein, 3-cyanopyridine hydrate is used as a raw material, and nicotinamide is prepared by catalysis of nitrile hydratase-containing microorganisms.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defect of high content of byproducts in the existing nicotinamide microbe catalytic production process, the invention constructs the nitrile hydratase mutant coding gene, and thalli obtained by the recombinant engineering bacteria after induction expression are used as enzyme sources, so that the recombinant engineering bacteria can be applied to nicotinamide production and the generation of byproduct nicotinic acid is reduced.
In order to solve the technical problems, the invention provides the following technical scheme:
a preparation method of a recombinant bacterium for expressing a nitrile hydratase mutant comprises the following specific steps:
(1) the method is characterized in that pET28-NHase plasmid of escherichia coli with high nitrile hydratase yield is used as a template, and an error-prone PCR method is adopted to construct a gene random mutation plasmid library, and the specific method is as follows:
recovering DNA products from the amplified fragments by agarose gel electrophoresis;
carrying out enzyme digestion on pET-28(a) idle body by using BamHI and HindIII, carrying out enzyme digestion on the recovered DNA product by using the same method, connecting the DNA fragment after enzyme digestion and pET-28(a) vector by using T4DNA ligase, and carrying out reaction at 16 ℃ overnight;
transforming the ligation product to DH10B competent cells with an electric converter, coating the competent cells on an LB plate of 50mg/mL kanamycin sulfate, culturing overnight, collecting thalli and extracting plasmids to obtain a gene random mutation plasmid library;
(2) the plasmid library is transferred into BL21 competent cells by a heat shock transformation method, the competent cells are coated on an LB plate containing kanamycin with the final concentration of 50mg/ml, a single clone is selected for bacterial detection, the target band is 1.9kb, and the bacterial sample system is as follows: (screening results graph)
(3) Randomly selecting bacteria-detecting positive monoclonal, inoculating into 48-well plate, shake culturing at 37 deg.C and 220rpm in 1ml liquid LB culture medium containing 50mg/ml kanamycin at final concentration for 16 hr, inoculating the culture solution into 50ml liquid LB culture medium containing 50mg/ml kanamycin at 3% volume ratio, shake culturing at 37 deg.C and 220rpm to OD600＝0.4～0.6, adding IPTG with the final concentration of 100umol/L and cobalt chloride with the final concentration of 40mg/L, performing shake culture at 18 ℃ and 220rpm for 24 hours to obtain fermentation liquor, directly adding 3-cyanopyridine with the final concentration of 10% into the fermentation liquor, reacting at 20 ℃ for 30 minutes, and immediately placing on ice for 30 minutes. Eliminating colonies with crystal precipitation;
(4) the strains obtained by screening are shaken again according to the method, and the concentration of the 3-cyanopyridine is increased to 20 percent or 30 percent to obtain the high-activity strains.
A recombinant bacterium for expressing a nitrile hydratase mutant is prepared by the preparation method of the recombinant bacterium for expressing the nitrile hydratase mutant.
Preferably, the recombinant bacteria contain a nitrile hydratase wild-type or mutant gene sequence including, but not limited to, one of SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO. 5.
Preferably, the nitrile hydratase wild type or mutant gene is expressed to synthesize a nitrile hydratase wild type or mutant, the amino acid sequence of which includes, but is not limited to, one of SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9, SEQ NO. 10.
A preparation method of nicotinamide uses the recombinant bacteria expressing the nitrile hydratase mutant as an enzyme source to produce nicotinamide, and comprises the following specific steps:
adding the thalli into pure water for resuspension, preserving heat at 20 ℃, using a 70 wt% concentration 3-cyanopyridine aqueous solution as a reaction substrate, feeding at an initial flow rate of 40g/min, reducing the flow rate according to the content of 3-cyanopyridine in a reaction solution in the reaction process, continuously reacting at 20 +/-2 ℃ to obtain nicotinamide, and reducing the byproduct nicotinic acid by more than 90%.
Preferably, the enzyme activity final concentration of the nitrile hydratase mutant in the reaction liquid is 70U/mL, the pure water is adjusted to pH8.0 +/-0.2 by using liquid alkali, the reaction substrate is prepared by double distilled water, the temperature of the reaction substrate is kept in a water bath at 50 ℃, and the 3-cyanopyridine final concentration is 30 wt% of the reaction substrate.
The invention has the following beneficial effects:
1. the invention uses the genome of Escherichia coli with high nitrile hydratase yield as a template, adopts error-prone PCR technology to recombine and construct a random mutant plasmid library, screens nitrile hydratase mutant genes from the library, and can efficiently induce and express nitrile hydratase mutant proteins after the recombinant genes are transformed into engineering bacteria.
2. The nitrile hydratase mutant protein is used as a catalytic enzyme, can efficiently catalyze a substrate 3-cyanopyridine to synthesize nicotinamide, and can remarkably reduce the generation of a byproduct nicotinic acid.
3. The recombinant engineering bacteria of the high expression nitrile hydratase mutant coding gene are induced and expressed to obtain thallus which is used as an enzyme source, hydration reaction is carried out under a proper condition to produce nicotinamide, and the byproduct nicotinic acid is over 90 percent lower.
Drawings
FIG. 1 is a diagram showing the amino acid sequence alignment of mutant strains and wild strains;
FIG. 2 shows the results of enzyme activity measurement of a nitrile hydratase mutant of a wild-type strain;
FIG. 3 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 1;
FIG. 4 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 2;
FIG. 5 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 3;
FIG. 6 shows the results of enzyme activity measurement of the nitrile hydratase mutant of the high-activity mutant strain 4;
wherein, the peak appearing in 5.5-6 min is nicotinamide in the reaction solution, and the peak appearing in 11min is byproduct nicotinic acid.
Detailed Description
The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.
Example 1: the recombinant engineering bacteria of the coding gene of the high-expression nitrile hydratase mutant is constructed according to the following method:
(1) the method for constructing the gene random mutation plasmid library by adopting the error-prone PCR method by taking pET28-NHase plasmid of escherichia coli with high nitrile hydratase yield as a template comprises the following steps:
recovering DNA products from the amplified fragments by agarose gel electrophoresis;
carrying out enzyme digestion on pET-28(a) idle body by using BamHI and HindIII, carrying out enzyme digestion on the recovered DNA product by using the same method, connecting the DNA fragment after enzyme digestion and pET-28(a) vector by using T4DNA ligase, and carrying out reaction at 16 ℃ overnight;
transforming the ligation product to DH10B competent cells by an electric converter (Bio-rad MicroPulser), coating the competent cells on an LB plate of 50mg/mL kanamycin sulfate, culturing overnight, collecting thalli and extracting plasmids to obtain a gene random mutation plasmid library;
(2) the plasmid library was transferred into BL21(DE3) competent cells by heat shock transformation, plated on LB plates containing kanamycin to a final concentration of 50mg/ml, and single colonies were selected for bacterial screening, with a target band of 1.9kb, and bacterial specimen lines as follows:
(3) randomly selecting bacteria-detecting positive monoclonal, inoculating into 48-well plate, shake culturing at 37 deg.C and 220rpm in 1ml liquid LB culture medium containing 50mg/ml kanamycin at final concentration for 16 hr, inoculating the culture solution into 50ml liquid LB culture medium containing 50mg/ml kanamycin at 3% volume ratio, shake culturing at 37 deg.C and 220rpm to OD600When the concentration is 0.4-0.6, adding IPTG with the final concentration of 100umol/L and cobalt chloride with the final concentration of 40mg/L, performing shake culture at 18 ℃ and 220rpm for 24 hours to obtain fermentation liquor, directly adding 3-cyanopyridine with the final concentration of 10% into the fermentation liquor, reacting at 20 ℃ for 30 minutes, and immediately placing on ice for 30 minutes. Eliminating colonies with crystal precipitation, wherein if the crystal precipitation is the 3-cyanopyridine, the reaction is not complete;
(4) the strains obtained by screening are shaken again according to the method, and the concentration of the 3-cyanopyridine is increased to 20 percent or 30 percent to obtain the high-activity strains.
In the embodiment, 4 high-activity mutant strains are obtained, a thallus sample of the high-activity mutant strains is subjected to enzyme activity to screen out partial strains, and hydration small-scale test verification is carried out, wherein the specific operations are as follows:
the collected thallus is used as an enzyme source, and the enzyme activity is measured by the following specific detection method:
1. the reaction system contained 0.5mL of 50mM ammonium chloride-ammonia buffer (pH8.0) containing 100mM 3-cyanopyridine.
2. Adding proper amount of thallus. The reaction was stirred at 25 ℃ for 60min and quenched by the immediate addition of 0.5mL of anhydrous acetonitrile. Centrifuge at 12000rpm for 1 min.
HPLC method used Agilent high performance liquid chromatography (Agilent 1100, USA), the column was Varianpursuit C18 reverse phase column (4.6 mM. times.250 mM), the mobile phase was 15mM potassium phosphate buffer (pH2.8), acetonitrile 92:8(v/v), the flow rate was set to 0.5mL/min, UV detector, and the detection wavelength was 265 mM.
4. Definition of nitrile hydratase enzyme activity: 1 unit (U) is the amount of enzyme required for catalyzing the formation of 1 mu mol nicotinamide at 25 ℃ for 1min, the unit is U/mL, the result is shown in Table 1, and an HPLC detection chart is shown in FIGS. 2-6:
TABLE 1 measurement results of enzyme Activity of fungal enzyme sources
| Bacterial strains | Enzyme activity (U/mL) |
| Wild type (SEQ NO.1) | 70 |
| Mutant strain 1(SEQ NO.2) | 119 |
| Mutant 2(SEQ NO.3) | 125 |
| Mutant 3(SEQ NO.4) | 107 |
| Mutant 4(SEQ NO.5) | 143 |
Respectively taking thalli of the 1 wild type and 4 high-activity mutant strains as enzyme sources, adding a proper amount of thalli into 3.5ml of pure water (adjusting the pH value to 8.0 +/-0.2 by liquid caustic soda), keeping the final enzyme activity concentration at 70U/ml, keeping the temperature at 20 ℃, and adding 1.5ml of 3-cyanopyridine in batches (the final concentration is 30 percent, and the water bath at 50 ℃): 150 ul/time/15 min, reacting for 1.5h after the addition is finished, carrying out post-treatment on the reaction liquid at 20 ℃, detecting the content of byproduct nicotinic acid, screening out recombinant engineering bacteria (the nicotinic acid is reduced by more than 90%), centrifuging the fermentation liquid at 4 ℃, 6000rpm for 10min, and collecting thalli, wherein the data is as follows:
TABLE 2 content of nicotinic acid as by-product in catalytic reaction solution of different strains
| Bacterial strain | Temperature/. degree.C | Nicotinic acid ppm |
| Wild type (SEQ NO.1) | 20 | 109.5 |
| Mutant strain 1(SEQ NO.2) | 20 | 9.3 |
| Mutant 2(SEQ NO.3) | 20 | 10.2 |
| Mutant 3(SEQ NO.4) | 20 | 9.8 |
| Mutant 4(SEQ NO.5) | 20 | 7.1 |
40L of reaction system: the collected thalli is taken as an enzyme source (the final enzyme activity concentration is 70U/ml), added into 20.5kg of pure water (the pH value is adjusted to be 8.0 +/-0.2 by liquid caustic soda), the temperature is kept at 20 ℃, 19.5kg of 70 wt% of 3-cyanopyridine aqueous solution is taken as a reaction substrate (pure water preparation and 50 ℃ water bath temperature keeping), the initial flow rate is 40g/min for feeding, the final 3-cyanopyridine concentration of the reaction solution is 30 wt%, the reaction is carried out at 20 +/-2 ℃, the reaction solution is subjected to post-treatment at 20, 30, 40 and 50 ℃ after the reaction is finished, and the byproduct nicotinic acid is reduced by more than 90% in the final nicotinamide product.
TABLE 3 Effect of different aftertreatment temperatures on nicotinic acid content
(5) Sequencing the recombinant strain obtained by screening to obtain nitrile hydratase gene, wherein the sequencing results of the wild type and mutant gene sequences are shown in SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4 and SEQ NO 5; the amino acid sequences corresponding to the wild type and the mutant gene sequences are shown in SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9 and SEQ NO 10; comparing the nitrile hydratase amino acid sequences in the mutant strain and the wild strain, the amino acid residue mutation sites of the mutant strain are mainly L49F, G deletion at 65, N108I, L131Q, E155V, V180L and P181T (the protein primary sequence comparison result is shown in figure 1), and comparing the enzyme activity determination result (the specific result is shown in figures 2-6), when the amino acid residues at the sites are mutated, the nitrile hydratase activity is still kept at a high position, and the generation of a byproduct, namely nicotinic acid, is obviously reduced.
In conclusion, the invention uses the genome of Escherichia coli with high nitrile hydratase yield as a template, adopts error-prone PCR technology to recombine and construct a random mutant plasmid library, screens nitrile hydratase mutant genes from the library, and can efficiently induce and express nitrile hydratase mutant proteins after the recombinant genes are transformed into engineering bacteria. The nitrile hydratase mutant protein is used as a catalytic enzyme, can efficiently catalyze a substrate 3-cyanopyridine to synthesize nicotinamide, and can remarkably reduce the generation of a byproduct nicotinic acid. The recombinant engineering bacteria of the high expression nitrile hydratase mutant coding gene are induced and expressed to obtain thallus which is used as an enzyme source, hydration reaction is carried out under a proper condition to produce nicotinamide, and the byproduct nicotinic acid is over 90 percent lower.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
SEQUENCE LISTING
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Claims (6)
1. A preparation method of a recombinant bacterium for expressing a nitrile hydratase mutant is characterized by comprising the following specific steps:
(1) the method is characterized in that pET28-NHase plasmid of escherichia coli with high nitrile hydratase yield is used as a template, and an error-prone PCR method is adopted to construct a gene random mutation plasmid library, and the specific method is as follows:
recovering DNA products from the amplified fragments by agarose gel electrophoresis;
the carrier pET-28(a) is digested with BamHI and HindIII, the recovered DNA product is digested with the same method, the digested DNA fragment and the carrier pET-28(a) are linked with T4DNA ligase, and the mixture is reacted at 16 ℃ overnight;
transforming the ligation product to DH10B competent cells by an electric converter (Bio-rad MicroPulser), coating the competent cells on an LB plate of 50mg/mL kanamycin sulfate, culturing overnight, collecting thalli and extracting plasmids to obtain a gene random mutation plasmid library;
(2) transferring the gene random mutation plasmid library into BL21 competent cells by a heat shock transformation method, coating the competent cells on an LB plate containing kanamycin with the final concentration of 50mg/ml, selecting a single clone for bacterial detection, wherein the target band is 1.9kb, and the bacterial sample system is as follows:
(3) randomly selecting bacteria to detect positive monoclonal, inoculating into 1mL liquid 48-pore plate LB culture medium containing 50mg/mL kanamycin at the final concentration, carrying out shake culture at 37 ℃ and 220rpm for 16h, inoculating the culture solution into 50mL liquid LB culture medium containing 50mg/mL kanamycin at 3% volume ratio, carrying out shake culture at 37 ℃ and 220rpm until OD is achieved600When the concentration is 0.4-0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 100umol/L and cobalt chloride with the final concentration of 40mg/L, performing shake table induction culture at 18 ℃ and 220rpm for 24 hours to obtain fermentation liquor, directly adding 3-cyanopyridine with the final concentration of 10% into the fermentation liquor, reacting at 20 ℃ for 30 minutes, immediately placing on ice for 30 minutes, and eliminating colonies precipitated by crystals;
(4) the strains obtained by screening are shaken again according to the method, and the concentration of the 3-cyanopyridine is increased to 20 percent or 30 percent to obtain the high-activity strains.
2. A recombinant bacterium for expressing a nitrile hydratase mutant, which is characterized in that: the recombinant bacterium expressing a nitrile hydratase mutant according to claim 1.
3. A recombinant bacterium which expresses a nitrile hydratase mutant according to claim 2, wherein: containing a nitrile hydratase wild-type or mutant gene sequence including, but not limited to, one of SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO. 5.
4. A recombinant bacterium which expresses a nitrile hydratase mutant according to claim 3, wherein: the nitrile hydratase wild type or mutant gene is expressed to synthesize a nitrile hydratase wild type or mutant, and the amino acid sequence of the hydratase wild type or mutant includes, but is not limited to, one of SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9, and SEQ NO. 10.
5. A nicotinamide preparation method is characterized in that nicotinamide is produced by using the recombinant bacteria expressing the nitrile hydratase mutant as claimed in any one of claims 2-4 as an enzyme source, and the specific steps are as follows:
adding the thalli into pure water for resuspension, preserving heat at 20 ℃, using 70 wt% of 3-cyanopyridine aqueous solution as a reaction substrate, feeding at an initial flow rate of 40g/min, reducing the flow rate according to the content of 3-cyanopyridine in a reaction solution in the reaction process, continuously reacting at 20 +/-2 ℃ to obtain nicotinamide, and reducing the byproduct nicotinic acid by more than 90%.
6. A process for the preparation of nicotinamide according to claim 5, which comprises: the enzyme activity final concentration of the nitrile hydratase mutant in the reaction liquid is 70U/mL, the pH of pure water is adjusted to 8.0 +/-0.2 by adopting liquid caustic soda, the reaction substrate is prepared by double distilled water, the temperature is preserved by water bath at 50 ℃, and the 3-cyanopyridine final concentration is 30 wt% of the reaction substrate.
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| WO2004056990A1 (en) * | 2002-12-19 | 2004-07-08 | Mitsui Chemicals, Inc. | Novel nitrile hydratase |
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| CN106148310A (en) * | 2016-08-11 | 2016-11-23 | 南京工业大学 | A kind of nitrilase mutants and the application in prepared by nicotinic acid thereof |
| CN107881163A (en) * | 2017-09-29 | 2018-04-06 | 浙江大学 | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides |
| CN108486088A (en) * | 2018-02-14 | 2018-09-04 | 浙江工业大学 | Nitrilase mutants and its application |
| CN109593750A (en) * | 2019-01-16 | 2019-04-09 | 江南大学 | A kind of nitrile hydratase variant, the genetic engineering bacterium containing the mutant and its application |
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| WO2004056990A1 (en) * | 2002-12-19 | 2004-07-08 | Mitsui Chemicals, Inc. | Novel nitrile hydratase |
| JP2008228628A (en) * | 2007-03-19 | 2008-10-02 | Mitsubishi Rayon Co Ltd | Method for producing nitrile hydratase |
| CN106148310A (en) * | 2016-08-11 | 2016-11-23 | 南京工业大学 | A kind of nitrilase mutants and the application in prepared by nicotinic acid thereof |
| CN107881163A (en) * | 2017-09-29 | 2018-04-06 | 浙江大学 | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides |
| CN108486088A (en) * | 2018-02-14 | 2018-09-04 | 浙江工业大学 | Nitrilase mutants and its application |
| CN109593750A (en) * | 2019-01-16 | 2019-04-09 | 江南大学 | A kind of nitrile hydratase variant, the genetic engineering bacterium containing the mutant and its application |
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