Patent Grant
11492606
This is a U.S. national stage application of PCT Application No. PCT/CN2019/074110 under 35 U.S.C. 371, filed Jan. 31, 2019 in Chinese, claiming priority of Chinese Application No. 201810136409.4, filed Feb. 9, 2018, all of which are hereby incorporated by reference.
The present invention relates to the technical field of biological engineering, specifically relates to a nitrilase mutant with improved catalytic activity and stereoselectivity and its application in the synthesis of chiral intermediate of pregabalin.
Nitrilase (Nitrilase EC 3.5.5.1) is a biocatalyst that catalyzes the hydrolysis of nitrile compound (R-CN) to produce carboxylic acid and ammonia, which is widely applied in the synthesis of (chiral) amino acid, carboxylic acid and their derivatives. Enzymatic hydrolysis of nitrile is appreciated in the academic circle and the industrial circle due to its advantages of mild reaction conditions, efficient process, friendly environment and high chemoselectivity, regio selectivity and stereo selectivity, etc.
Due to that wild-type nitrilase is generally difficult to adapt to the demands of industrial environment, improving the catalytic performance of nitrilase by means of rational and non-rational protein modifications has become a research hotspot. DeSantis et al use point saturation mutation technology to modify wild-type nitrilase, and the obtained mutant A190H is able to catalyze 3 M 3-hydroxyglutaronitrile to produce (R)-4-cyano-3-hydroxybutyrate with the product ee value of up to 99% (J. Am. Chem. Soc., 2003,125:11476-11477). Schreiner et al utilize PCR technology to modify nitrilase AtNIT2 from Arabidopsis thaliana, and the activity of the obtained nitrilase towards benzyl cyanide hydrolysis is improved by 4 times (ChemCatChem, 2010, 2:263-267).
Pregabalin, of which the chemical name is (3S)-3-aminomethyl-5-methylhexanoic acid, is a first-line drug that treats neuropathic pain, epilepsy, anxiety and nervous centralis pain caused by spinal cord injury, trauma or multiple sclerosis etc. Compared with traditional drugs of the similar type, pregabalin has the advantages of low dose, low administration times, long duration, less side effect and high tolerance etc., which has become one of the best-selling drugs in the world.
Construction of the chiral center is the key to pregabalin synthesis, research on synthesis of pregabalin chiral intermediate through biocatalysis is getting more and more attention. Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. However, the catalytic activity of the nitrilase in the prior art is low (J. Mol. Catal. B: Enzym. 2006, 41:75-80), and is difficult to satisfy the requirement of industrial application. Therefore, developing nitrilase mutant construction technology and obtaining nitrilase of high activity plays an important role for efficient manufacturing of pregabalin.
The purpose of the present invention is to modify wild-type nitrilase by means of genetic engineering, in a bid to achieve significant improvement of catalytic performance of the modified nitrilase such as catalytic activity and stereoselectivity and meet the requirements of industrial application.
In order to realize such objects, the technical solution as follows is adopted in the present invention:
A nitrilase mutant, of which the amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3.
In the present invention, the amino acid sequences of crucifer turnip (Brassica rapa) nitrilase (BrNIT) and (Arabidopsis thaliana) arabidopsis nitrilase (AtNIT) are respectively displaced, especially the multiple sites of amino acid in the area of sites 225-285 are displaced to obtain the nitrilase mutant as shown in SEQ ID NO.1 or SEQ ID NO.3. Research proved that compared with wild-type nitrilase, the catalytic activity and stereoselectivity of the mutant to the substrate racemic IBSN were significantly improved.
Conservative substitutions of other amino acid sites of the nitrilase, addition or deletion of one or a few amino acids, N-terminal truncations and C-terminal truncations, such mutant forms also fall in the scope of the present invention.
The present invention further provides a coding gene that encodes the nitrilase mutant, of which the nucleotide sequence is as shown in SEQ ID NO.2 or SEQ ID NO.4.
The present invention further provides a recombinant vector containing the coding gene. Preferably, the original vector is pET28b.
The present invention further provides a recombinant strain containing the recombinant vector. The recombinant vector is introduced into the host cell to obtain the recombinant genetic engineering strain. The host cell can be various normal host cells in this field. Preferably, the host cell is Escherichia coli BL21.
Another object of the present invention is to provide a method for preparing the nitrilase mutant, comprising the steps as follow:
(1) based on turnip nitrilase gene or arabidopsis nitrilase gene sequence, designing PCR primer. Using Arabis alpina L. cDNA as a template, utilizing the primer for amplification to obtain DNA fragment I or DNA fragment II that contains sites 673-855 of the Arabis alpina L. nitrilase nucleotide sequence;
(2) taking the recombinant plasmid that carries turnip nitrilase gene or arabidopsis nitrilase gene sequence as a template, utilizing reverse PCR amplification to obtain the BrNIT plasmid fragment lack in sites 676-858 of turnip nitrilase nucleotide sequence or obtain the AtNIT plasmid fragment lack in sites 673-855 of arabidopsis nitrilase nucleotide sequence;
(3) recombining DNA fragment I with BrNIT plasmid fragment or recombining DNA fragment II with AtNIT plasmid fragment, and introducing the recombinant product into the host bacteria to obtain a nitrilase mutant expression strain;
(4) conducting induced expression of the nitrilase mutant strain to obtain the nitrilase mutant.
Crucifer turnip (Brassica rapa) nitrilase (BrNIT), arabidopsis (Arabidopsis thaliana) nitrilase (AtNIT) and Arabis alpina L. (Arabis alpine) nitrilase (AaNIT) all have the activity of catalyzing racemic IBSN to produce pregabalin chiral intermediate (S)-3-cyano-5-methylhexanoic acid. However, they have different advantages and disadvantages in terms of catalytic activity and stereoselectivity. In the present invention, the turnip nitrilase BrNIT and arabidopsis nitrilase AtNIT was taken as parents and using peptide fragment displacement method, the 226-286 peptide fragment of BrNIT amino acid sequence and 225-285 peptide fragment of AtNIT amino acid sequence were replaced with 225-285 peptide fragment of Arabis alpina L. nitrilase AaNIT, to obtain nitrilase mutants BrNIT225-285 and AtNIT225-285, respectively, of which the amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3, in a bid to improve the catalytic activity and stereoselectivity of nitrilase.
The preferred plasmid in step (2) is pET28b.
The preferred PCR primer required for preparing nitrilase mutant BrNIT225-285 in step (1) for amplifying DNA fragment I is:
The preferred PCR primer required for amplifying BrNIT plasmid fragment in step (2) is:
The preferred PCR primer required for preparing nitrilase mutant AtNIT225-285 in step (1) for amplifying DNA fragment II is:
The preferred PCR primer required for amplifying AtNIT plasmid fragment in step (2) is:
Preferably, the host cell is Escherichia coli BL21.
Another object of the present invention is to apply the nitrilase mutant in catalyzing racemic IBSN to prepare (S)-3-cyano-5-methylhexanoic acid.
Specifically, the recombinant strains containing nitrilase mutant coding genes were taken as biocatalyst in the formation of immobilized wet cell, wet cell or the purified enzyme extracted from ultrasonication of the wet cell. Racemic IBSN is used as a substrate, the reaction medium is used as buffer solution with pH 5.0-10.0, and the reaction is performed at 25-45° C. and 100-300 rpm. After the reaction is completed, the reaction mixture containing (S)-3-cyano-5-methylhexanoic acid is obtained, which was further separated and purified.
Preferably, in the reaction system, the final concentration of the substrate is 0.5-1.5 M, the used wet cell content is 10-30 g/L. Further preferably, the additive amount of the wet cells required for conversion of 1 mol substrate is 20 g.
Preferably, the reaction medium is Tris-HCl buffer solution with pH 8.0.
Preferably, the hydrolysis reaction is conducted at 30-35° C., 200 rpm.
Preferably, the applied wet cell is recombinant Escherichia coli BL21(DE3)/pET28b-BrNIT225-285 or E. coli BL21(DE3)/pET28b-AtNIT225-285 containing nitrilase mutant coding gene. The fermentation culture is as follows: the recombinant Escherichia coli containing nitrilase mutant coding gene is inoculated in LB culture medium containing kanamycin and cultured until OD600 reaches 0.6-0.8, after which isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM is added to induce the protein expression at 28° C. for 10-12 hours. After centrifugation, the cells were collected.
Compared with the previous invention, the present invention has the following benefits:
(1) The present invention provides a directed enzyme modification method, which displaces the critical peptide fragment of the turnip nitrilase or arabidopsis nitrilase with the critical peptide fragment of Arabis alpina L. nitrilase, to construct nitrilase mutant with improved activity and stereoselectivity, which has good application prospect in efficient catalyzing racemic IBSN to synthesize (S)-3-cyano-5-methylhexanoic acid.
(2) The activity of the nitrilase mutant BrNIT225-285 catalyzing the hydrolysis of racemic IBSN is 2.5 times of that of wild type and the enantiomeric ratio (E value) is increased from 200 to 500 or above; while the activity of AtNIT225-285 catalyzing racemic hydrolysis of IBSN is 1.9 times of that of wild type. The stereoselectivity is improved without compromising catalytic activity, which satisfies the requirement of industrial application.
Below is further description of the invention in conjunction with embodiments, however, the scope of protection of the present invention is not limited to these embodiments only.
The sources of the main experimental materials of the following embodiments:
Escherichia coli host strains E. coli BL21(DE3) and E. coli BL21(DH5α) were purchased from Transgen; the expression vector pET-28b(+) was purchased from Novagen; Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme; 2xTsingKe Master Mix(blue) were purchased from TsingKe; kanamycin was purchased from Takara Bio (Dalian); IPTG is product of Promega.
1. Construction of Nitrilase Mutants
The present invention adopts a simple, quick and efficient seamless DNA cloning technology (ClonExpress®) to conduct directed cloning of the amplified peptide fragment into the BrNIT plasmid fragment that is missing the corresponding gene segment.
Through comparing and analyzing the nucleotide sequence and amino acid sequence of crucifer nitrilase, the peptide fragments were determined. The amino acid sequence of the wild type turnip nitrilase was SEQ ID No.5, this protein was coded by the nucleotide sequence of SEQ ID No.6; the amino acid sequence of the wild type Arabis alpina L. nitrilase was SEQ ID No.7, this protein was coded by the nucleotide sequence of SEQ ID No.8.
The AaNIT nucleotide sequence was taken as template for cloning peptide fragment sites 0-85, 85-175, 175-225, 225-285 and 285-342, respectively.
Meanwhile, the recombinant plasmid containing BrNIT nucleotide sequence was taken as a template and the corresponding primers were designed to amplify the BrNIT plasmid fragment that is missing 0-86, 86-176, 176-226, 226-286 and 286-342 peptide fragment.
The plasmid containing BrNIT sequence that is missing the corresponding peptide fragment was linearized, and the forward/reverse PCR primer 5′ for amplifying the inserting fragment was introduced with the terminal sequence of the linearized plasmid, allowing the terminals of PCR primers 5′ and 3′ respectively bear sequences consistent to the two terminals of the linearized vector. The primer sequences were as shown in Table 1.
The PCR amplification for the peptide fragments was conducted as follows: PCR reaction system (50 μL): Template DNA<1 μg, 2×TsingKe® Master Mix, forward and reverse primers at 0.2 μM respectively, ddH2O was supplemented to the total volume. The PCR progress was performed at 94° C. for 5 minutes, followed by 30 cycles of 94° C. for 30 seconds, 58° C. for 30 seconds and 72° C. for 10 seconds, after which, the reaction was performed at 72° C. again for 10 minutes. The amplified products went through agarose gel electrophoresis analysis, excising recovery, inactivated at 65° C. for 10 minutes, and placed at 4° C. for use.
Vector linearization was achieved through reverse PCR amplification. PCR reaction system (50 μL) was as follows: template DNA 0.1 ng-1 ng, 2×Phanta Max Buffer, dNTPs (10 mM each) 0.2 mM, forward and reverse primers at 0.2 μM respectively, Phanta Max Super-Fidelity DNA Polymerase 1 U, ddH2O was supplemented to the total volume. The PCR progress was performed at 95° C. for 30 seconds, followed with 30 cycles of 95° C. for 15 seconds, 63° C. for 15 seconds and 72° C. for 6.0 minutes, after which the reaction was performed at 72° C. for 5 minutes. The amplified products went through agarose gel electrophoresis analysis, excising recovery, inactivated at 65° C. for 10 minutes, and placed at 4° C. for use.
NanoDrop™ One/OneC ultramicro-UV spectrophotometer was used to calculate the concentration of the above obtained insert fragments and linearized vectors. The additive amounts of the linearized vectors were calculated for the insert peptide fragments and the corresponding missing peptide fragments. Composition of the ligation reaction system was shown in Table 2. The PCR sample was mixed and placed at 37° C. for 30 minutes, then reduced to 4° C.
10 μL recombinant product was added into 100 μLE. coli BL21(DH 5α) competent cell, which was sprayed onto LB plate with 50 mg/L kanamycin. The plates were cultured at 37° C. for 10-12 h. A single colony was picked into LB fluid medium with 50 mg/L kanamycin for plasmid extraction. The positive colonies were transformed into E. coli BL21(DE3) competent cell and cultured overnight to obtain nitrilase mutant expression strain.
2. Nitrilase Mutant Gene Expression
A single colony was picked and placed into 5 mL LB fluid medium with kanamycin at a final concentration of 50 mg/L The cultivation was performed at 37° C. and 200 rpm for 6-8 hours. The above seed solution was transferred to fresh LB fluid medium containing 50 mg/L kanamycin at 2% volume ratio, which was also cultured at 37° C. and 150 rpm. Until the OD600 of the cell culture reached about 0.6-0.8, IPTG (final concentration at 0.1 mM) was added to induce the gene expression at 28° C. and 150 rpm for 10-12 hours. The cultured cells were collected and centrifuged at 4° C. and 8000 rpm for 10 minutes, washed twice with normal saline and centrifugated again. The obtained cells were disrupted, separated and purified, which was further stored at −20° C. The electrophoresis diagram of the obtain nitrilase mutant BrNIT225-285 was as shown in
3. Determine the Activity of Recombinant Escherichia coli Containing Nitrilase Mutant
The activity of the recombinant Escherichia coli containing nitrilase mutant (E. coli BL21(DE3)/pET28b-BrNIT0-85, E. coli BL21(DE3)/pET28b-BrNIT85-175, E. coli BL21(DE3)/pET28b-BrNIT175-225, E. coli BL21(DE3)/pET28b-BrNIT225-285 and E. coli BL21(DE3)/pET28b-BrNIT285-342) were determined. The reaction was performed in Tris-HCl buffer solution (50 mM, pH 8.0) containing nitrilase mutant (10 mL), racemic IBSN 30 g/L, wet cells 10 g/L at 30° C. and 200 rpm for 15 minutes. After reaction, 500 μL of reaction sample was taken and added with 200 μL 2 M HCl to end reaction.
The enantiomeric excess value of the substrate racemic IBSN and the product 3-cyano-5-Methylhexanoic acid was determined by gas chromatography. The gas chromatograph model was 7890N (Agilent) and the capillary column model was BGB-174 (BGB Analytik Switzerland). Chromatographic condition: injection volume was 1.0 μL, the temperatures of both the injection port and the detector were 250° C., the column temperature was 120° C. maintaining for 15 minutes, then the temperature was raised from 10° C./min to 170° C. and maintain for 9 minutes. The carrier gas was high-purity helium, the flow rate was 1.0 mL/min, the split ratio was 50:1.
The calculation of the enantiomeric excess value (ee) and the conversion rate (c) was referred to the calculation method of Rakels et al (Enzyme Microb. Technol., 1993, 15:1051).
The activities of the nitrilase mutants were as shown in Table 3:
escherichia
coli containing nitrilase mutant
E. coli BL21(DE3)/pET28b-BrNIT
E. coli BL21(DE3)/pET28b-BrNIT0-85
E. coli BL21(DE3)/pET28b-BrNIT85-175
E. coli BL21(DE3)/pET28b-BrNIT175-225
E. coli BL21(DE3)/pET28b-BrNIT225-285
E. coli BL21(DE3)/pET28b-BrNIT285-342
4. Comparison of Nitrilase Mutant BrNIT225-285 and Wild Type Nitrilase in Catalyzing Racemic IBSN Hydrolysis
The recombinant E. coli BL21(DE3)/pET28b-BrNIT225-285 obtained from culture and the recombinant E. coli BL21(DE3)/pET28b-BrNIT containing wild type nitrilase were taken as biocatalysts, compare the effect of their stereoselectivity in hydrolyzing racemic IBSN.
Reaction system composition (100 mL): Tris-HCl buffer solution (50 mM, pH 8.0), 1.5 g wet cell and 10 g racemic IBSN. The reaction was conducted at 35° C. and 200 rpm and 500 μL sampled was taken every 1 hours, which was further added with 200 μL 2 M HCl to stop the reaction. The progresses of the mutant and wild type nitrilase in catalyzing racemic IBSN hydrolysis were shown in
5. Biosynthesis of (S)-3-Cyano-5-Methylhexanoic Acid with Recombinant E. coli BL21(DE3)/pET28b-BrNIT225-285
The biosynthesis of (S)-3-cyano-5-methylhexanoic acid was performed in 100 mL Tris-HCl buffer solution (pH 8.0) with 2.0 g wet cells of recombinant E. coli BL21(DE3)/pET28b-BrNIT225-285 (final concentration at 20 g/L) and 1 M racemic IBSN (136 g/L). The reaction was conducted at 30° C. and 200 rpm for 8 h, and during which, 500 μL sample was taken every 1 hours, which was further added with 200 μL 2 M HCl to stop the reaction. The sample test method was in reference to Step 3. The conversion rate reached 39.8%, and the ee value of the product (S)-3-cyano-5-methylhexanoic acid exceeded 99.3%. Compared with reported catalytic process, the additive amount of the cells was reduced by 2.5 times.
Construction of arabidopsis nitrilase mutant AtNIT225-285 and its application in synthesizing (S)-3-cyano-5-methylhexanoic acid.
The amino acid sequence of the wild type arabidopsis nitrilase was SEQ ID No.9, which was encoded by the nucleotide sequence of SEQ ID No.10.
Arabidopsis nitrilase mutant AtNIT225-285 was constructed in reference to Embodiment 1. The primers used for mutant construction were as shown in Table 4.
The recombinant E. coli BL21(DE3)/pET28b-BrNIT225-285 containing arabidopsis nitrilase mutant and the recombinant E. coli BL21(DE3)/pET28b-BrNIT containing wild type arabidopsis nitrilase were obtained in reference to Embodiment 1. After induced expression, whole cell was collected and disrupted, separated and purified, to obtain nitrilase mutant AtNIT225-285. The electrophoresis diagram was shown in
The reaction was performed in 10 mL Tris-HCl buffer solution (50 mM, pH 8.0) with 0.1 g (wet weight) resting cells containing arabidopsis nitrilase mutant and wild type nitrilase and 0.3 g racemic IBSN at 30° C. and 200 rpm. The activity of mutant AtNIT225-285 in catalyzing racemic IBSN was 1.9 times of that of wild type nitrilase. After reaction for 24 hours, the conversion of IBSN by wild type AtNIT and mutant AtNIT225-285 reached 25.64% and 48.76%, respectively, both of which the ee value exceeded 98.5%.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810136409.4 | Feb 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/074110 | 1/31/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/154249 | 8/15/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7361507 | Pierrard | Apr 2008 | B1 |
| Number | Date | Country |
|---|---|---|
| 104962540 | Oct 2015 | CN |
| 105176955 | Oct 2015 | CN |
| Entry |
|---|
| Xie, Zhiyi et al. “Cloning and Optimization of a Nitrilase for the Syntheisis of (3S)-3-Cyano-5-Methyl hexanoic Acid”, Journal of Molecular Catalysis B: Enzymatic, vol. 41, Jun. 5, 2006, pp. 75-80. |
| Number | Date | Country | |
|---|---|---|---|
| 20210009981 A1 | Jan 2021 | US |
