CN111154746B

CN111154746B - Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid

Info

Publication number: CN111154746B
Application number: CN202010032055.6A
Authority: CN
Inventors: 郑仁朝; 刘长丰; 吴哲明; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2021-10-08
Anticipated expiration: 2040-01-13
Also published as: CN111154746A

Abstract

The invention discloses an amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid, belonging to the technical field of enzyme engineering. The amidase mutant is obtained by carrying out single-site mutation or multi-site mutation on 378 th position, 402 th position or 403 th position of an amino acid sequence shown in SEQ ID NO. 1. Compared with parent strain, the amidase mutant provided by the invention has greatly improved activity, and the activity of the reaction enzyme is still kept in a higher state when a crude extract of the amidase or whole cells of engineering bacteria are used for catalysis. In addition, the amidase mutant provided by the invention can adapt to the catalytic temperature of 30-55 ℃, and lays a foundation for the industrial enzymatic production of 2-chloronicotinic acid.

Description

Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to an amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid.

Background

2-chloronicotinic acid is an important intermediate compound for synthesizing pesticides and medicines. In the field of pesticides, 2-chloronicotinic acid can be used for synthesizing a series of bactericidal active compounds of sulfonylurea herbicides nicosulfuron, amide herbicides diflufenican, amide bactericides boscalid, triazolone and the like; in the field of medicine, 2-chloronicotinic acid is an important intermediate for synthesizing anti-AIDS drugs, namely nevirapine, antidepressant drugs, mirtazapine, non-steroidal anti-inflammatory analgesics, namely niflumic acid, pranoprofen, nicotinylmethacic acid and the like. Due to the wide application of 2-chloronicotinic acid in pesticides and medicines, the market demand is increasing.

At present, the production method of 2-chloronicotinic acid is mainly a chemical synthesis method. For example, 2-chloronicotinic acid is synthesized by taking nicotinic acid as a starting material through nitrogen oxidation, chlorination and hydrolysis; 3-cyanopyridine is used as a raw material, and is subjected to nitrogen oxidation, chlorination and hydrolysis to synthesize 2-chloronicotinic acid; 2-chloro-3-methylpyridine oxidation; a cyclization method using ethyl cyanoacetate as a starting material; a chlorination hydrolysis method using 2-chloro-3-trifluoromethylpyridine as a starting material.

Since Lonza, Switzerland, applied for the synthesis of 2-chloronicotinic acid in 1977 (US4144238), the chemical industry such as Japan organic synthesis and Japan Photogrong chemical was first and then introduced into the research on the synthesis of 2-chloronicotinic acid (JP 59144759; JP 56169672). At present, the 3-cyanopyridine method is mainly adopted in the industry to produce the 2-chloronicotinic acid, but the production process has the defects of heavy pollution, long synthesis steps, low yield and the like.

Amidases are a class of hydrolases that catalyze the synthesis of the corresponding carboxylic acid from amide compounds. Because of their advantages such as high stereoselectivity and broad substrate spectrum, the biosynthesis of (chiral) carboxylic acids, (chiral) amide derivatives and optically pure amino acids using amidases as biocatalysts is increasingly gaining attention from researchers. Amidase hydrolysis of 2-chloronicotinamide has become an important method for the synthesis of 2-chloronicotinic acid (CN 101857889; Bioorganic Chemistry,2018,76: 81-87).

The development of technologies such as protein engineering, crystal structure analysis and the like provides a powerful tool for enzyme molecule modification. Among the reported amidases, the Pantoea-derived amidase Pa-Ami mutant G175A constructed by Zhengrenzhao et al can efficiently catalyze 2-chloronicotinamide to synthesize 2-chloronicotinic acid (CN 201710974605.4).

The construction of the amidase biocatalyst with high catalytic activity is of great significance for realizing the high-efficiency and green production of the 2-chloronicotinic acid.

Disclosure of Invention

The invention aims to further carry out molecular modification on a Pantoea (Pantoea sp.) amidase Pa-Ami mutant G175A by using a site-directed saturation mutagenesis technology, provide a mutant protein, improve the hydrolysis activity of the mutant protein on 2-chloronicotinamide, and facilitate the application of the amidase in the preparation of 2-chloronicotinic acid.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention further obtains the mutant with improved enzyme activity by utilizing a semi-rational design method on the basis of the Pantoea amidase Pa-Ami mutant G175A (the amino acid sequence is shown as SEQ ID NO. 1). The amidase mutant is obtained by carrying out single-site mutation or multi-site mutation on 378 th position, 402 th position or 403 th position of an amino acid sequence shown in SEQ ID NO. 1.

Specifically, the single-site mutation is as follows: replacing alanine (a) at position 378 of the amino acid sequence shown in SEQ ID No.1 with valine (V) or threonine (T); or replacing valine (V) at position 402 of the amino acid sequence shown in SEQ ID NO.1 with leucine (L); or replacing leucine (L) at position 403 of the amino acid sequence shown in SEQ ID NO.1 with valine (V) or threonine (T).

The multi-site mutation is a combination of the single-site mutations. Preferably, the amidase mutant is: the amino acid sequence shown in SEQ ID NO.1 has the amino acid sequence that the alanine at the 378 position is mutated into threonine and the leucine at the 403 position is mutated into valine, or the alanine at the 378 position is mutated into threonine and the valine at the 402 position is mutated into leucine, or the alanine at the 378 position is mutated into valine and the leucine at the 403 position is mutated into valine, or the alanine at the 378 position is mutated into valine and the valine at the 402 position is mutated into leucine and the leucine at the 403 position is mutated into valine.

Compared with a parent amidase Pa-Ami mutant G175A, the amidase mutant has greatly improved activity and is suitable for the catalytic temperature of 30-55 ℃.

The invention also provides a coding gene for coding the amidase mutant and a recombinant genetic engineering bacterium containing the coding gene.

Preferably, the coding gene is cloned to an expression vector pET28b and transformed into a host cell Escherichia coli BL21 to obtain a recombinant gene engineering bacterium.

The invention also aims to provide application of the amidase mutant in catalyzing 2-chloronicotinamide to prepare 2-chloronicotinic acid.

The application comprises the following steps: taking wet thalli obtained by fermenting and culturing engineering bacteria containing amidase mutant coding genes or enzyme extracted after the wet thalli is crushed as a biocatalyst, taking 2-chloronicotinamide as a substrate, taking a buffer solution with the pH of 7.5-8.5 as a reaction medium, carrying out conversion reaction at the temperature of 30-60 ℃ and under the condition of 150-500 r/min, and taking reaction liquid for separation and purification after the reaction is finished to obtain the 2-chloronicotinic acid.

The amidase mutant can be used in the form of whole cells of engineering bacteria, crude enzyme without purification, and enzyme protein after partial purification or complete purification. If necessary, the amidase mutant of the present invention can also be used in the form of immobilized enzyme or immobilized cell using an immobilization technique known in the art.

In the reaction system, the initial concentration of the substrate is 50-300 mM, and the substrate is fed to the final concentration of 50-200 mM every 10-60 min. The dosage of the catalyst is 0.25-2.5 g/L based on the dry weight of the thalli.

Preferably, the initial concentration of substrate is 200mM, and a final concentration of 100mM 2-chloronicotinamide is added each time less than 20% of substrate remains.

The amount of the catalyst used was 2.5g/L in terms of cell mass (dry cell weight).

Preferably, the reaction medium is Tris-HCl buffer solution with pH value of 8.0, and the reaction conditions are 40 ℃ and 200 r/min.

The wet thallus of the invention is prepared by the following method: inoculating the engineering bacteria containing the coding gene of the amidase mutant into LB culture medium containing 50mg/L kanamycin at the final concentration, culturing for 12h at 37 ℃ at 150r/min, then transferring the engineering bacteria into fresh LB culture medium containing 50mg/L kanamycin at the final concentration by 1% of the inoculation amount by volume concentration, culturing at 37 ℃ at 150r/min until the thallus concentration OD600 is 0.4-0.8, then adding IPTG (preferably 0.1mM) at the final concentration of 0.1-1 mM into the culture medium, inducing and culturing for 12h at 28 ℃ at 150r/min, taking the culture for centrifugation, and collecting the precipitate to obtain wet thallus. The LB liquid medium consists of (g/L): peptone 10, yeast extract 5, NaCl 10, pH 7.0; LB plate medium composition (g/L): peptone 10, yeast extract 5, NaCl 10, agar 15, solvent deionized water, pH 7.0.

The invention has the following beneficial effects:

compared with parent strain, the amidase mutant provided by the invention has greatly improved activity, and the activity of the reaction enzyme is still kept in a higher state when a crude extract of the amidase or whole cells of engineering bacteria are used for catalysis. In addition, the amidase mutant provided by the invention can adapt to the catalytic temperature of 30-55 ℃, and lays a foundation for the industrial enzymatic production of 2-chloronicotinic acid.

Drawings

FIG. 1 shows the progress of the feeding reaction of G175A and triple mutant A378V/V402L/L403V in the preparation of 2-chloronicotinic acid by whole-cell catalysis of 2-chloronicotinamide (initial concentration of 200 mM).

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

The parent amidase adopted in the specific embodiment is a Pantoea sp (Pantoea sp.) source amidase Pa-Ami (GenBank NO. WP008109374) mutant G175A constructed at the earlier stage of the subject group, and is disclosed in Chinese patent application literature (CN201710974605.4), wherein the amino acid sequence of the amidase is shown as SEQ ID NO.1, and the nucleotide sequence of the amidase is shown as SEQ ID NO. 2.

Example 1 Induction and expression purification of amidase

The method comprises the following steps: cultivation of recombinant bacteria

Inoculating 10 μ L of glycerol tube stock solution into 10mL of liquid LB culture medium (containing Kan 50 μ g/mL), culturing at 37 deg.C for 12 hr at 200r/min, transferring to 100mL of fresh LB culture medium containing Kan at an inoculum size of 2%, and culturing to OD₆₀₀To reach 0.6, IPTG was added to a final concentration of 0.1mM and incubation was induced overnight (12h) at 28 ℃. After completion of the culture, the cells were collected by centrifugation and washed 2 times with 0.85% physiological saline.

Step two: purification of amidases

(1) 5g of wet thalli are suspended in 50mL of Tris-HCl buffer solution (20mM, pH 8.0), shaken uniformly, and then cells are crushed by an ultrasonic cell crusher (the crushing power is 40W, 5s of ultrasonic work is carried out each time, the interval is 5s, and 99 cycles are totally performed);

(2) after the crushing is finished, taking the crushing liquid, centrifuging at the temperature of 4 ℃ for 20min at the speed of 12,000r/min, and removing cell fragments;

(3) collecting the supernatant (crude enzyme solution) for subsequent separation and purification of the enzyme;

(4) the purification column is Ni-NTA, the column volume is 20mL, the column volume is 5-10 times of the column volume loading balance buffer (20mM Tris-HCl,300mM NaCl and 50mM imidazole, pH 8.0) for balance, and the flow rate is 1.5 mL/min;

(5) loading at a rate of 1.5 mL/min;

(6) eluting with a loading equilibration buffer to remove unadsorbed protein;

(7) eluting with elution buffer (20mM Tris-HCl,300mM NaCl, and 250mM imidazole, pH 8.0) to collect the target protein;

(8) the enzyme solution containing the target protein was dialyzed overnight (2 buffer changes) against 20mM Tris-HCl, pH 8.0.

(9) The protein concentration of the dialysate was determined spectrophotometrically using a bradfold protein concentration kit.

Example 2 site-directed saturation mutagenesis and screening of amidases

Description of site-directed saturation mutagenesis reference (Applied Microbiology and Biotechnology,2014,98(6): 2473-. The specific process is as follows:

the method comprises the following steps: site-directed mutagenesis

The amino acids at the 378 th, 402 th and 403 th sites in the amino acid sequence of the parent pantoea amidase mutant G175A are subjected to saturation mutation, primers A378, V402 and L403 (see Table 1) are designed, and plasmid pET28-G175A which clones the gene encoding pantoea amidase mutant G175A is used as a template for full plasmid amplification.

TABLE 1 design table of site-directed saturation mutagenesis primers

Note: n is A/G/C/T, K is G/T, and M is A/C.

The PCR system is as follows: 2 XPhata Max buffer 25. mu.L, dNTP mix (10mM) 1. mu.L, mutant primers 10. mu.M each 1. mu.L as shown in Table 1, plasmid pET28-Pa-Ami 0.5. mu.L, Phanta Max DNA polymerase 0.5. mu.L, ddH₂And O is supplemented to 50 mu L.

The PCR condition is pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 65 ℃ for 15s, extension at 72 ℃ for 6.5min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃. The PCR product was analyzed by 0.9% agarose gel electrophoresis, 20. mu.L of the PCR product was taken, 1. mu.L of Dpn I was added, the digestion was carried out at 37 ℃ for 3 hours to remove the template plasmid DNA, and the inactivation was carried out at 65 ℃ for 10 min.

Step two: transformation of amidase mutants

Coli BL21(DE3) was added to 10. mu.L of the PCR product from step one, allowed to stand on ice for 30min, heat-shocked at 42 ℃ for 90s, added to 600. mu.L of LB medium without kanamycin, incubated at 37 ℃ for 1h at 180r/min, spread on LB plates containing kanamycin (50mg/L), and incubated overnight at 37 ℃.

Step three: cell culture and high throughput screening

Single colonies were picked and inoculated into 96-well plates (containing 1mL of LB medium containing 50mg/L kanamycin), and cultured at 37 ℃ and 150r/min to OD₆₀₀About 0.5, then IPTG was added to a final concentration of 0.1mM and cultured at 28 ℃ for 12 hours. Taking a new 96-well culture plate, respectively adding 100 μ L of cultured bacteria liquid one by one, taking 2-chloronicotinamide and hydroxylamine hydrochloride as double substrates, and FeCl₃The solution is used as a color developing agent, the G175A engineering bacteria cells are used as a reference, the judgment is carried out according to the speed of color change (yellow green → dark red) of the color development, and strains with the color change speed higher than that of a control group are selected as primary screening positive bacteria.

Example 3 rescreening of amidase Positive mutants

The positive cells obtained in example 2 were cultured under the culture conditions of example 1 to obtain mutant whole cells. Weighing wet thalli, adding buffer solution, and uniformly beating to prepare bacterial suspension with wet cell concentration of 40 g/L. The reaction system for determining the activity of the mutant is as follows: total system 10mL, final cell concentration of 0.25g/L, 50mM Tris-HCl (pH 8.0), 50mM 2-chloronicotinamide. Oscillating at 55 deg.C and 150r/min, reacting for 10min, adding 100 μ L2M hydrochloric acid into 1mL to terminate the reaction, diluting by 10 times, and detecting with liquid chromatography. Liquid chromatography detection conditions: mobile phase: acetonitrile: water: phosphoric acid 250: 750: 1, the flow rate is 1mL/min, and the detection wavelength of 2-chloronicotinic acid is 270 nm.

Definition of enzyme activity unit (U): the cells required to catalyze the production of 1. mu. mol of 2-chloronicotinic acid per minute from 2-chloronicotinamide at 55 ℃ and pH 8.0 are used as a viable unit (U).

And extracting plasmids from the mutant with the whole-cell enzyme activity higher than G175A for sequencing. DNA sequencing of positive clones with improved activity shows that alanine (codon GCA) at position 378 is replaced by valine (codon GTA) or threonine (codon ACA), valine (codon GTG) at position 402 is replaced by leucine (codon CTG), leucine (codon CTG) at position 403 is replaced by valine (codon GTG) or threonine (codon ACG), and amidase mutant engineering bacteria E.coli BL21(DE3)/pET28-A378V, E.coli BL21(DE3)/pET28-A378T, E.coli BL21(DE3)/pET28-V402L, E.coli BL21(DE3)/pET28-L403V, E.coli BL21(DE3)/pET28-L403T are obtained. The enzyme activity of the mutant is detected in Table 2.

TABLE 2 Whole cell enzyme Activity of Positive mutants

Mutants	Whole cell enzyme activity (U/g)
		G175A	1100±57
A378V	1489±50
		A378T	1357±47
V402L	1810±65
		L403V	1427±34
L403T	1650±43

Example 4 construction of iterative mutation library and Activity assay

The forward single mutant of example 3 was subjected to iterative mutation, primers were designed (see Table 3), and transformation was performed after whole plasmid amplification (see Table 4) according to the first step of example 2 using plasmid of amidase mutant as a template. Single colonies were picked and cultured in 10mL LB tubes containing kanamycin, sequenced and deposited. The recombinant bacteria with iterative mutation were cultured in the culture manner described in step one of example 1, and the whole-cell enzyme activity was measured in the manner described in example 3. The whole-cell enzyme activity and the sequence of each mutant are shown in Table 5, wherein the enzyme activity of the three mutants A378V/L403V/V402L reaches 2786U/g (wet cells) which is 2.53 times of that of the original strain.

TABLE 3 primers for iterative mutation

TABLE 4 amplification of iterative mutations

TABLE 5 Whole cell enzyme Activity of iterative mutants

Example 5 determination of the specific enzyme Activity of the mutants

Pure enzyme was prepared from the recombinant bacteria with improved enzyme activity in example 3 and example 4 in the same manner as in example 1, and the specific enzyme activity of the amidase mutant was determined. Specific enzyme activity assay system (10 mL): taking appropriate amount of enzyme solution to react for 10min under 50mM 2-chloronicotinamide and 50mM (pH 8.0) Tris-HCl buffer solution, terminating the reaction with 2M hydrochloric acid, diluting the sample by 10 times, and performing high performance liquid chromatography detection.

Specific enzyme activity unit (U) definition: the amount of enzyme required to catalyze the formation of 1. mu. mol of 2-chloronicotinic acid per minute from 2-chloronicotinamide at 55 ℃ and pH 8.0 is taken as one activity unit (U).

As can be seen from Table 6, the specific enzyme activity of the triple mutant A378V/L403V/V402L is 127.44U/mg, which is 2.4 times that of G175A.

TABLE 6 specific enzyme Activity of the mutants on 2-chloronicotinamide

Example 6 amidase mutant A378V/V402L/L403V full-cell catalysis of 2-chloronicotinamide to synthesize 2-chloronicotinic acid

2-Chloronicotinib was prepared using amidase triple mutant E.coli BL21(DE3)/pET28-A378V/V402L/L403V cells obtained in example 3 as a catalyst and 2-chloronicotinamide as a substrate. The reaction system is as follows: 200mM Tris-HCl buffer (pH 8.0), initial concentration of 200mM 2-chloronicotinamide, 1.25g/L cells (dry cell weight), at 40 degrees C, 200r/min under reaction. Every time when the substrate residue was less than 20%, 100mM of 2-chloronicotinamide was added, and 8 feeding was performed in total, and the final cumulative total amount of 2-chloronicotinic acid in the system was 930 mM. G175A accumulated only 570mM 2-chloronicotinic acid under the same conditions. The feeding reaction process is shown in figure 1.

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Claims

1. An amidase mutant which is obtained by single-site mutation or multi-site mutation at position 378, 402 or 403 of an amino acid sequence shown in SEQ ID NO.1,

wherein, the amidase mutant with single-point mutation is that the 378 th alanine of the amino acid sequence shown in SEQ ID NO.1 is mutated into valine or threonine, or the 402 th valine is mutated into leucine, or the 403 th leucine is mutated into valine or threonine;

the multi-site mutated amidase mutant is the amino acid sequence shown in SEQ ID NO.1, wherein the 378 th alanine is mutated into threonine and the 403 th leucine is mutated into valine, or the 378 th alanine is mutated into threonine and the 402 th valine is mutated into leucine, or the 378 th alanine is mutated into valine and the 403 th leucine is mutated into valine, or the 378 th alanine is mutated into valine and the 402 th valine is mutated into leucine and the 403 th leucine is mutated into valine.

2. A gene encoding the amidase mutant of claim 1.

3. A recombinant genetically engineered bacterium comprising the coding gene of claim 2.

4. The use of the amidase mutant as claimed in claim 1 for catalyzing 2-chloronicotinamide to prepare 2-chloronicotinic acid.

5. The use of claim 4, comprising: taking wet thalli obtained by fermenting and culturing engineering bacteria containing amidase mutant coding genes or enzyme extracted after the wet thalli is crushed as a biocatalyst, taking 2-chloronicotinamide as a substrate, taking a buffer solution with the pH of 7.5-8.5 as a reaction medium, carrying out conversion reaction at the temperature of 30-60 ℃ and under the condition of 150-500 r/min, and taking reaction liquid for separation and purification after the reaction is finished to obtain the 2-chloronicotinic acid.

6. The use according to claim 5, wherein the substrate is initially fed to the reaction system at a concentration of 50 to 300mM and is fed to the reaction system at intervals of 10 to 60min to a final concentration of 50 to 200 mM.

7. The use according to claim 5, wherein the amount of the catalyst used in the reaction system is 0.25-2.5 g/L based on the dry weight of the cells.

8. The use according to claim 5, wherein the reaction medium is Tris-HCl buffer at pH 8.0 and the reaction temperature is 40 ℃.