CN116064443A - Imine reductase mutant and application thereof - Google Patents
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Abstract
The invention provides an imine reductase mutant and application thereof in (S) -nornicotine synthesis, in particular to a method for preparing (S) -nornicotine with high chiral purity by reducing myosmine by using the obtained mutant, wherein the imine reductase mutant from Myxococcus furlvus has high activity, high reaction substrate concentration, high reaction yield and high optical purity of a product, is simple to operate in the reaction process, has low energy consumption, meets the green chemical requirements, and can be applied to biotransformation preparation of (S) -nornicotine compounds.
Description
Technical Field
The invention belongs to the field of biocatalysis, and relates to a method for producing (S) -nornicotine by reducing myosmine (CAS: 532-12-7) with optical purity of >99% by using imine reductase from myxococcus and using the mutant as biocatalyst and NADP (H) as coenzyme.
Background
(S) -Nornicotine, also known as (S) -Nornicotine, english name (S) - (-) -Nornicotine, CAS number: 494-97-3, the molecular formula C9H12N2 is as follows:
(S) -nornicotine is also a potential useful compound precursor in the treatment of Parkinson ' S disease, alzheimer ' S disease and Tourette ' S syndrome [ Viswanaf A, joseph L, [ J ] ACS comb. Sci.2017,19,286-298] in the prior study of nicotine hapten in synthetic nicotine vaccines and in the prior study of nicotine addiction.
The synthesis method of the (S) -nornicotine mainly comprises chemical synthesis or biological enzyme catalysis synthesis, the synthesis of the (S) -nornicotine by using the chemical synthesis method needs multi-step reaction, chiral derivatization reagent or metal catalyst and the like can be used in the process, the conditions are harsh, environmental pollution is easy to cause, the optical purity of the obtained product is low, 98.0% or more is difficult to reach, the yield is low, and a plurality of limitations are provided in actual mass production [ Charles H.M., steven J.Q ] [ J ] Journal of Medicinal Chemistry,2017,19,286-298]. Therefore, it is important to search for a more green and efficient bio-enzyme catalytic method.
The report of biocatalysis synthesis of (S) -norsmoke is less, in 2021, raymond McCague and the like catalyze 2-pyridine-1-pyrroline to generate (S) -2- (3-pyridine) -pyrrolidine to generate (S) -nornicotine by using imine reductase from sources, the substrate concentration reaches 0.4M/L (58.4 g/L), the substrate concentration is improved, and the conversion rate is obviously reduced [ US 10913962.B2].
Because of the unique property of amine substrates, the concentration of the substrates cannot be too high in the reduction process, otherwise, the reaction conversion rate is obviously reduced, and the consumption of NAD (P) H of expensive coenzyme is also higher, so that searching for imine reductase with higher activity and high substrate tolerance becomes a key factor for efficiently reducing imine substrate 2-pyridine-1-pyrroline to generate (S) -nornicotine.
Disclosure of Invention
In order to efficiently synthesize (S) -nornicotine under mild reaction conditions, the invention performs directed evolution on imine reductase MfIR (WP_ 074958336.1) from Myxococcus fulvus to obtain an imine reductase mutant with improved enzyme activity and substrate tolerance, and reduces imine to prepare (S) -2- (3-pyridine) -pyrrolidine with high chiral purity.
In a first aspect, the invention provides a method for synthesizing (S) -nornicotine with high chiral purity, wherein an engineered imine reductase is used as a catalyst, the engineered imine reductase has at least 90% identity with SEQ ID NO.1, and the ee value of the mutant conversion product (S) -nornicotine is >99%.
In another aspect, there is provided an imine reductase mutein, said mutein being a non-natural protein and said mutein being an imine reductase mutant mutated at one or more positions corresponding to positions 65, 123 and 212 in SEQ ID NO. 1:1-291:
in another preferred embodiment, asparagine (N) at position 65 is mutated to valine (V), proline (P), preferably valine (V).
In another preferred embodiment, threonine at position 123 is mutated to proline (P), tyrosine (Y), phenylalanine (F), valine (V), preferably proline (P).
In another preferred embodiment, methionine (M) at position 212 is mutated to valine (V), leucine (L) and isoleucine (I), preferably isoleucine (I).
More specifically, the following combinatorial mutations: mutation at position 65 to valine (V) and mutation at position 212 to leucine (L) or isoleucine (I); mutation at position 123 to proline (P), tyrosine (Y) and mutation at position 212 to leucine (L) or isoleucine (I).
In a third aspect, the imine reductase mutant of the second aspect catalyzes the following reaction: specifically, the catalytic reaction takes a culture, a bacterial cell or a crushing liquid of fermented genetically engineered bacteria containing an imine reductase mutant as a catalyst, wheat ston as a substrate, NADP (H) as a coenzyme, and a buffer solution with pH of 6.0-10.0 as a reaction medium, and the catalytic reaction is carried out under the conditions of 25-50 ℃ and stirring;
the reaction has one or more characteristics selected from the group consisting of:
(i) The reaction system contains the thallus with the amount of 10-50g/L, more preferably 10-30g/L;
(ii) The pH of the reaction system is 6.0 to 10.0, preferably 6.0 to 8.0, more preferably 7.5;
(iii) The reaction system temperature is 20 ℃ to 50 ℃, preferably 20 ℃ to 30 ℃, more preferably 25 ℃.
(iv) The concentration of the catalytic substrate is 5-150g/L, more preferably, the concentration of the substrate is 80-130g/L;
(v) The ee value of the (S) -nornicotine obtained by catalysis is more than or equal to 99.0 percent.
Drawings
FIG. 1 shows the results of the induced expression of MfIR and mutant proteins. Wherein M represents Marker,1 is wild type MsIR1 soluble protein expression, and 2-6 is representative mutant soluble protein expression.
FIG. 2 HPLC profile of nornicotine racemate.
FIG. 3 HPLC profile of (S) -nornicotine.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention.
As used herein, the term "AxxB" means that amino acid a at position xx is changed to amino acid B, e.g. N65V means that amino acid N at position 65 is mutated to V, and so on.
In a preferred embodiment of the present invention, the preparation method of the imine reductase mutant of the present invention is as follows: coli as an expression host. Specifically, the preparation method comprises the following steps: (1) The gene of the corresponding mutation site of the imine reductase MsIR1 is constructed on a pET28a expression vector to obtain a recombinant plasmid with the target enzyme gene. (2) The recombinant plasmid is transferred into a host bacterial cell, preferably escherichia coli BL21 (DE 3), and the corresponding engineering strain is obtained. (3) Inoculating engineering strain to seed culture medium for culturing, inoculating to fermentation culture medium according to a certain proportion, culturing for a certain period, and adding inducer IPTG or lactose or their mixture for inducing culture for a certain period. And (4) centrifugally collecting thalli, and carrying out high-pressure crushing.
EXAMPLE 1 construction of library of imine reductase MfIR mutants
A simulated protein structure of MsIR1 is obtained by taking a protein (PDB ID:6to 4) with 64% of sequence homology with the MsIR1 as a template and carrying out homologous modeling, non-conserved residues in a substrate binding pocket of the simulated protein structure are selected to carry out saturation mutation respectively, degenerate codon NNK is adopted to design a mutation primer, and pET28a-MsIR1 is taken as the template. The obtained monoclonal colony is picked into a 96-well deep pore plate for culture, and the expressed protein is subjected to high-throughput activity screening by detecting the decrease of NADPH at 340 nm. The specific method of the reaction is as follows: the substrate concentration is 100mM,NADPH 0.25mg/mL, the crude enzyme solution is 20 mu L, the sodium phosphate buffer is filled to 200 mu L, the enzyme label instrument detects the decrease of NADPH at 340nm, if the enzyme activity is relatively high, the consumption of NADPH is rapid, and the slope of the decrease curve is large.
The sites for library-building mutations were 13, 65, 95, 120, 121, 123, 124, 127, 171, 174, 175, 178, 179, 212, 216, 236, 241 and 242, respectively, and the useful mutation sites for achieving increased enzyme activity were 65, 123 and 212, respectively, mutants 1-8.
EXAMPLE 2 construction of the library of imine reductase MfIR combination mutants
According to the saturated mutation result, selecting sites 123 and 212 with obviously improved activity, constructing a two-point combined mutant library, and obtaining mutants 9, 10, 11 and 12 with further improved activity, wherein the protein sequence is shown as SEQ ID NO. 2-5. The conversion rate and the stereoselectivity of the mutant are examined by establishing a conversion reaction for a substrate, the concentration of the substrate is 100mM, bacterial liquid is not concentrated, the detection is carried out for 2 hours, and the results of enzyme activity measurement and conversion reaction are shown in Table 1.
TABLE 1 enzyme Activity of MfIR and its mutants, conversion to substrate and stereoselectivity
Example 3: induction expression of imine reductase mutants
50mL of seed solution is prepared, the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L and NaCl 10 g/L), a single colony of the genetically engineered bacterium is picked up by an inoculating loop and inoculated into the culture medium, and the culture is carried out at 37 ℃ and 200rpm for overnight. The seed solution cultured overnight was transferred to the fermentation medium at 1% of the inoculum size, and cultured at 37℃and 200rpm to OD 600 About 0.6-0.8 mM IPTG is added, induction is carried out for more than 10 hours, bacterial cells are collected by centrifugation culture solution at 8000rpm, and high-pressure crushing is carried out, thus obtaining crude enzyme liquid of imine reductase. SDS-PAGE electrophoresis pattern shows the induction expression condition of MsIR1 and mutant, as shown in figure 1, wherein M represents Marker,1 is the expression supernatant of MfIR wild type protein, 2-6 is the expression supernatant of mutant 2-6 in turn, and the result shows that the expression amount of mutant protein obtained by the method of the embodiment is unchanged from the expression amount of wild type soluble protein.
EXAMPLE 4 wild type Whole cell catalytic Synthesis of (S) -nornicotine Using imine reductase
10ml of phosphate buffer (pH 7.5), 30-40mg/ml of thalli, 2eq of glucose, NADP + 0-0.5mg/ml, GDH powder 10mg, substrate concentration 10-90mg/ml,28℃and TLC plate to determine the progress of the reaction. After 24 hours, adding sodium hydroxide saturated solution to adjust the pH value to more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
EXAMPLE 5 catalytic Synthesis of (S) -nornicotine Using crude enzyme solution of imine reductase wild type
10ml of phosphate buffer (pH 7.5), 30-40mg/ml of crude enzyme solution of imine reductase bacterial cells, 2eq glucose, NADP + 0-0.5mg/ml, GDH powder 10mg, substrate concentration 10-90mg/ml,30℃and TLC plate to determine the progress of the reaction. After 24 hours, adding sodium hydroxide saturated solution to adjust the pH value to more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
| Substrate concentration | Coenzyme NADP + | Conversion rate | ee | |
| 1 | 10 | 0 | 21.6 | 99.7 |
| 2 | 10 | 0.2 | 99.2 | 99.8 |
| 3 | 20 | 0.2 | 97.8 | 99.7 |
| 4 | 30 | 0.3 | 94.5 | 99.8 |
| 5 | 30 | 0.4 | 99.3 | 99.8 |
| 6 | 40 | 0.4 | 99.2 | 99.7 |
| 7 | 50 | 0.4 | 83.4 | 99.7 |
| 8 | 50 | 0.5 | 89.4 | 99.7 |
| 9 | 60 | 0.5 | 88.5 | 99.7 |
| 10 | 70 | 0.5 | 76.3 | 99.7 |
| 11 | 80 | 0.5 | 64.9 | 99.7 |
EXAMPLE 6 Whole cell catalyzed Synthesis of (S) -nornicotine Using imine reductase
Phosphate buffer 10ml, thallus 25mg/ml, glucose 1.2eq, NADP + 0.1mg/ml, GDH powder 10mg, substrate concentration 50mg/ml,28℃and TLC plates to determine the progress of the reaction. After 12 hours, adding sodium hydroxide saturated solution to adjust the pH value to more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
EXAMPLE 7 Whole cell catalytic Synthesis of (S) -nornicotine Using Iminium reductase mutant 11
10ml phosphate buffer (pH 7.5), 20mg/ml cell, 2eq glucose, 0.1mg/ml NADP + 10mg GDH powder, substrate concentration 50mg/ml,28℃reaction, TLC plate to determine the progress of the reaction. After 12 hours, adding sodium hydroxide saturated solution to adjust the pH value to more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
| Substrate concentration | Coenzyme NADP + | | ee | ||
| 1 | 10 | 0 | 21.6 | 99.7 | |
| 2 | 10 | 0.2 | 99.2 | 99.8 | |
| 3 | 40 | 0.2 | 99.4 | 99.7 | |
| 4 | 80 | 0.3 | 99.5 | 99.8 | |
| 5 | 120 | 0.3 | 99.3 | 99.8 | |
| 6 | 160 | 0.3 | 99.2 | 99.7 | |
| 7 | 120 | 0.4 | 99.3 | 99.7 | |
| 8 | 130 | 0.4 | 99.2 | 99.7 | |
| 9 | 140 | 0.4 | 99.5 | 99.7 | |
| 10 | 150 | 0.4 | 98.5 | 99.7 | |
| 11 | 160 | 0.4 | 90.6 | 99.7 |
Example 8: whole cell catalytic synthesis of (S) -nornicotine using imine reductase mutant 11
10ml phosphate buffer (pH 7.5), 30-40mg/ml thallus, 2eq glucose, 0-0.5mg/ml NADP + 10mg GDH powder, substrate concentration 10-90mg/ml,28℃reaction, TLC plate to determine the progress of the reaction. After 24 hours, adding sodium hydroxide saturated solution to adjust the pH value to more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
The implementation is as follows: catalytic synthesis of (S) -nornicotine using crude enzyme solution of imine reductase mutant 11
10ml of phosphate buffer (pH 7.5), 30-40mg/ml of crude enzyme solution of imine reductase bacterial cells, 2eq of glucose, 0-0.5mg/ml of NADP + 10mg GDH powder, substrate concentration 10-150mg/ml, reaction at 30℃and TLC plate to determine the progress of the reaction. After 24 hours, add sodium hydroxide to saturateAnd adjusting the pH value of the solution to above 10, centrifuging to remove denatured proteins, extracting the supernatant with dichloromethane, drying, spin-drying to collect the product, and detecting by HPLC.
Example 10: whole cell catalytic synthesis of (S) -nornicotine using imine reductase mutant 11
1L phosphate buffer (pH 7.5), imine reductase cell 30mg/ml,1.2eq glucose, NADP + 0.4mg/ml of GDH100mg of powder was fed with substrate in batches, the final concentration of substrate was 100mg/ml, reacted at 28℃and the progress of the reaction was judged by TLC plates. After the reaction is completed, adding a saturated solution of sodium hydroxide to adjust the pH value to be more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, and spin-drying to collect a product. Yield 96%, purity 98%, e.e. 99.8%.
Example 11: whole cell catalytic synthesis of (S) -nornicotine using imine reductase mutant 11
1L phosphate buffer (pH 7.5), imine reductase cell 30mg/ml,1.2eq glucose, NADP + 0.5mg/ml of GDH100mg of powder was fed with substrate in portions, the final concentration of substrate was 120mg/ml, reacted at 30℃and the progress of the reaction was judged by TLC plates. After the reaction is completed, adding a saturated solution of sodium hydroxide to adjust the pH value to be more than 10, centrifuging to remove denatured proteins, extracting supernatant with dichloromethane, drying, and spin-drying to collect a product. Yield 92%, purity 98%, e.e. 99.6%.
EXAMPLE 12 crude enzyme liquid catalyzed Synthesis of (S) -nornicotine Using imine reductase mutant 11
1L phosphate buffer (pH 7.5), 30mg/ml crude enzyme solution of imine reductase bacterial cells, 1.2eq glucose, NADP + 0.3mg/ml of GDH powder 100mg was fed with substrate in portions, the final concentration of substrate was 150mg/ml, reacted at 30℃and the progress of the reaction was judged by TLC plates. ReactionAfter completion, adding saturated solution of sodium hydroxide to adjust the pH value to more than 10, centrifuging to remove denatured protein, extracting supernatant with dichloromethane, drying, and spin-drying to collect the product. Yield 94%, purity 98.5%, e.e. 99.7%.
Sequence listing
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Ala Arg Lys Ala Gly His Gly Gln Asp Asp Phe Ser Val Leu Ala Arg
275 280 285
Phe Leu Arg
290
Claims (10)
1. An imine reductase mutein, characterized in that said mutein is mutated to valine at glutamine at position 65 corresponding to the amino acid sequence shown in SEQ ID No. 1; threonine at position 123 is mutated to proline, tyrosine, phenylalanine, valine; methionine at position 212 is mutated to valine, leucine or isoleucine.
2. An imine reductase mutein, characterized in that said mutein has a mutation at a position corresponding to the amino acid sequence shown in SEQ ID No.1: mutation at position 123 to proline or tyrosine and mutation at position 212 to leucine or isoleucine.
3. The imine reductase mutein-encoding gene according to claim 1 or 2.
4. An expression vector comprising a gene encoding the imine reductase mutein of claim 1 or 2.
5. A recombinant cell comprising a gene encoding the imine reductase mutein of claim 1 or 2.
6. Use of an imine reductase mutein according to claim 1 or 2 for the preparation of chiral (S) -nornicotine
7. The use according to claim 6, wherein said use is for the synthesis of (S) -nornicotine by catalytic reaction with Mastigmine (CAS: 532-12-7) as substrate.
8. The use according to claim 7, wherein the catalytic reaction is a reduction reaction at a reaction temperature of 20 ℃ to 50 ℃ with a wet cell obtained by fermentation culture of a genetically engineered bacterium encoding the imine reductase protein as a catalyst, with a substrate of myosmine (CAS: 532-12-7) and a buffer solution having a pH of 6.0 to 10.0 as a reaction medium.
9. The use according to claim 8, wherein in the catalytic reaction, the concentration of the catalytic substrate in the reaction system is 5-150g/L, the amount of the cells contained is 10-50g/L, the pH of the reaction system is 6.0-10.0, and the reaction temperature is 25-35 ℃.
10. The use according to claim 8, wherein NADP is added to the reaction system + Glucose, glucose dehydrogenase, and reacting at 150rpm-250rpm for 5-25 hours.
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