RU2833281C2 - Method of using reduction for producing (s)-nicotine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of producing (S)-nicotine by reduction. Said method involves carrying out a process for reducing an alkene compound, represented by formula I, and/or iminium cationic compound represented by formula II, to obtain (S)-nicotine, wherein reduction is carried out using a biocatalytic method or a chiral metal catalyst.

EFFECT: present invention enables to obtain (S)-nicotine, which does not require methylation and provides safety, as well as high output and high purity of the obtained product.

10 cl, 3 dwg, 2 tbl, 11 ex

Description

AREA OF TECHNOLOGY

The present invention relates to the field of organic synthesis technology and, more particularly, to a method for producing (S)-nicotine by reduction.

PREREQUISITES FOR THE CREATION OF THE INVENTION

(S)-Nicotine, also known as nicotine, is an alkaloid naturally occurring in plants belonging to the Solanaceae family and is a vital element in tobacco. Generally, tobacco leaves contain 1.5-3.5% (S)-nicotine, which is mainly obtained through extraction. Although chemical synthetic methods for obtaining nicotine have been described, such methods are still in the early stages of development and are more expensive than extraction processes. Chemical synthetic methods described so far include chemical cleavage, asymmetric hydrogenation, the use of chiral auxiliary methods, and so on.

In the publication of the patent document No. WO 2014174505, a method for synthesizing nicotine by catalyzing pseudooxynicotine with imine reductases (an enzyme that is a ketoreductase), specifically using imine reductases whose source is Streptomyces sp. GF3546 and Streptomyces sp. GF3587, is disclosed. It should be noted that the method detailed in the patent provides exclusively for the production of (R)-nicotine and does not provide instructions for the production of (S)-nicotine.

Patent document publication No. US10913962B discloses a method for synthesizing (S)-nicotine. The method uses catalysis of myosmin using an enzyme that is a reductase to obtain (S)-nornicotine, followed by reductive methylation of (S)-nornicotine. The method solves the problem of economic efficiency by using biological catalysis in obtaining (S)-nornicotine, which leads to a significant yield of (S)-nornicotine. In addition, formaldehyde is used as a methyl source in the methylation process, and formic acid serves as a reducing agent.

In order to overcome the disadvantages of the above methods, which either require methylation for the synthesis of (S)-nicotine or cannot produce (S)-nicotine by enzymatic catalysis without methylation, it is important to develop a method that eliminates the need for methylation in the production of (S)-nicotine.

ESSENCE OF THE INVENTION

The present invention provides a method for producing (S)-nicotine by reduction, in particular a method for producing (S)-nicotine by reduction.

The present invention provides a method for producing (S)-nicotine by reduction, and the method comprises:

carrying out a process of reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II, thereby obtaining (S)-nicotine:

Considering the limited production methods disclosed in the prior art, in which either methylation is required for the synthesis of (S)-nicotine or it is impossible to obtain (S)-nicotine through enzymatic catalysis without methylation, the present invention creatively develops a completely new method for efficiently producing (S)-nicotine without methylation, in other words, reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II. The method has the advantages of simple operations, safety and reliability, high yield and high purity.

In the present invention, the recovery specifically provides the following two solutions.

The recovery process is carried out using a biocatalytic method, and the biocatalytic method includes:

in a coenzyme cyclic system, catalytically reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II using imine reductase as a catalyst for a catalytic reduction reaction to thereby obtain (S)-nicotine.

Preferably, the coenzyme cycling system comprises a coenzyme, glucose and glucose dehydrogenase.

Preferably, the coenzyme comprises a NADP (nicotinamide adenine dinucleotide phosphate) salt and/or a NAD (nicotinamide adenine dinucleotide) salt, preferably a NADP salt.

Preferably, the glucose dehydrogenase comprises the amino acid sequence presented under SEQ ID No: 1.

SEQ ID No: 1:

MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTTNLTGAFLGSREAIKY FVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGR.

Preferably, the imine reductase comprises an amino acid sequence as set forth in SEQ ID No: 2-6, SEQ ID No: 8, SEQ ID No: 11, SEQ ID No: 12, or an amino acid sequence having at least 95% identity with the amino acid sequence as set forth in SEQ ID No: 12, preferably an amino acid sequence as set forth in SEQ ID No: 2-4, SEQ ID No: 12, or an amino acid sequence having at least 95% identity with the amino acid sequence as set forth in SEQ ID No: 12.

SEQ ID No:2:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGEAIAASDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARSLESWCHTRGARYLDGAILCFPDQIGTTDASIICSGASTAFSEAEPV LRLLAPPLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEVEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ.

SEQ ID No:3:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ.

SEQ ID No: 4:

MRHLSVIGLGAMGSALATTLIKGGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQQLLDEARDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPDQIGTSDA SIICSGASAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAL.

SEQ ID No: 5:

MRPISVIGLGAMGSALATTLLKAGHPVTVWNRSAAKATPLIALGAILAPSVSEAIAAGDITLICVDNYAVSQQLLDEASNAVTGKLVVQLSTGSPLGARTLESWCHARGACYLDGAILCFPDQIGTTDASIICSGANAAFREAEPV LRLLAPTLEHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLTDHAADAGIDNSFPRFAADLFEEGVEQGLGQQEVSALIKVLRARNGAAQ.

SEQ ID No: 6:

MRHLSVIGLGAMGSALATTLIKAGHPVTVWNRSAAKSAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQQLLDEASDAVAGKQLVQLSTGSPQGARALESWSHARGARYLDGAILCFPDQIGTSDASIICSGANTAFSDAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVTKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ.

SEQ ID No: 7:

MGTALVEAFLAGGHATTVWNRTPGKADGVVARGAVVAETVAEAVAASPLVVVCLWDDAVVRDVLHPVADALAGRVVVNLTNGTPAQAREMAAWAAEHGVEYVDGGIMAIPPGIGTEHAFVLYSGAEAAFEAHREVLER LGAAKYLGADAGLAALFDLALLSGMYGTFAGLWHSLAMVRTENVSAAEFVPMLGPWMQAMIGGNLDRLAHQLDTGDYGHEVVSNLAMQAAAFPNIVQASLDQGIRPDLMAPIQRLMDQAVAAGHGAEDVAVVVDLLKN.

SEQ ID No: 8:

MKPTLTVIGAGRMGSALIKAFLQSGYTTTVWNRTKAKSEPLAKLGAHLADTVRDAVKRSDIIVVNVLDYDTSDQLLRQDEVTRELRGKLLVQLTSGSPALAREQETWARQHGIDYLDGAIMATPDFIGQAECALLYSGSAALFEKH RAVLNVLGGATSHVGEDVGHASALDSALLFQMWGTLFGTLQALAISRAEGIPLEKTTAFIKLTEPVTQGAVADVLTRVQQNRLTADAQTLASLEAHNVAFQHLLALCEERNIHRGVADAMYSVIREAVKAGHGKDDFAILTRFLK.

SEQ ID No: 9:

MVSSPYLNVTAYPKVRNLPWPVPGPIRVASQILELRPMTTIGFLGAGRMGSALVKSLLEAGHSVHVWNRTAEKAQALADFGAVPEPSAERAAGPAEIVIVNLLDYEASDAELRKPDVAEALKGKLLVQLTSGSPKTARETGRWAGDHGIAYLDGAIMATPNFIGG AETVILYSGSKTHFEKHEGLFKALGGKSAFVGEDFGTASALDSALLSQMWGTLFGTLQALAVCRAEGIEHDVYAGFLMSAQPMIDGAQQDLMERIRDGRDLADAQTLATVAVHNVAFHHLRDLIADRDLNPAFGDALGSLLETALRNDHQDDDFAVLARFMGAK.

SEQ ID No: 10:

MTDLGKSAVTVLGLGAMGTALAEALLAAGHPTTVWNRSPARTAGPAQRGAAVAAATAEAIAASRLIVVCLLDHTSVHAVLDGQELTGRIVVNLTSGTPGQARELDARVAERGGDHLDGAVLAVPSMIGTPDASVLYSGSRGAFDTHR PVLEVFGAADYVGADPGAASLQDAALLSAMYGQVAGVLHAFALVRSAGVTATEFLPRLVGWLTAMGGFPADAARRIDARAYADDVDAALTMQVTAVRNLVRAAREQGVSAELIAPLVPVMQRRIDDGDGGDDLAALVEVITAEEVA.

SEQ ID No: 11:

MTDKPPVTVLGLGAMGTALARTLLNAGYPTTVWNRTASKTAPLTELGAHAADSPADAIARGELVLACLLDYDSVHQTLAGTGDALRGKAFVNLTNGTPEQARALAGKLDTAYLDGGIMAVPPMIGSPGAFLFYSGEIAVFEQYRP VLESFGEAIEVGTDPGLAALHDLALLSAMYGMFGGVLQAFALTGSAGVSAASLAPLLHRWLDGMSGFIAQSAAQLDSGDFATGVVSNLAMQDTGFANLFRAAKEQGISTGQLEPLGALIRRRVEDGHGAEDLAGIVEYLKIGANA.

SEQ ID No: 12:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ.

A person skilled in the art, using conventional methods, introduces mutations at one or more amino acids of the amino acid sequences presented under SEQ ID No: 2-12, thereby obtaining mutant variants of imine reductase. Mutant variants of imine reductase are screened for their ability to catalyze substrates necessary for the production of (S)-nicotine with a degree of conversion and an enantiomeric excess (ee) value corresponding to the working standards established for imine reductase.

Preferably, the amino acid sequence with at least 95% identity to the sequence of SEQ ID No: 12 shows the presence of mutations when compared with the amino acid sequence of SEQ ID No: 12 at amino acid residues in any one of the following positions or a combination of at least two of the following positions: L73, S148, V171 and A172.

Additionally preferably, the amino acid residue at position 73 is mutated to replace L with Q or V, the amino acid residue at position 148 is mutated to replace S with R, the amino acid residue at position 171 is mutated to replace V with Y, N, A or S, and the amino acid residue at position 172 is mutated to replace A with V or F.

Additionally preferably, the amino acid sequence with at least 95% identity to the sequence of SEQ ID No: 12 further demonstrates the presence of mutations when compared to the amino acid sequence of SEQ ID No: 12 at amino acid residues in any one of the following positions or a combination of at least two of the following positions: A57, A176, Y230 and S241.

Additionally preferably, the amino acid residue at position 57 is mutated to replace A with R, the amino acid residue at position 176 is mutated to replace A with G, the amino acid residue at position 230 is mutated to replace Y with G, A or T, and the amino acid residue at position 241 is mutated to replace S with G or A.

As an example, an amino acid sequence having at least 95% identity to the sequence of SEQ ID No: 12 may be as follows:

SEQ ID No: 13:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQVLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAAAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 14:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 15:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPGASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 16:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAAVAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPTASLKTWSAAIGRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 17:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAAVAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAIGRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 18:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAAAAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAIGRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No: 19:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LSLLAPTLDHVAEAVGAAAAQDCAVVAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAIGRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No:20:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ;

SEQ ID No:21:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAIARLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ or

SEQ ID No:22:

MRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIARSDITLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPV LRLLAPTLDHVAEAVGAAAAQDCAVAAYFGGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPTASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ.

A person skilled in the art, using conventional methods, obtains mutant variants of imine reductase with any combination of the above mutation points. The mutant variants of imine reductase are screened for their ability to catalyze substrates necessary for the production of (S)-nicotine with a conversion rate of 99% or greater and an ee value of 99% or greater.

Preferably, the catalytic reduction reaction is carried out at a temperature in the range of 15 to 45°C, such as 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C; other specific temperatures within this range may also be selected and are not described in detail herein.

Preferably, the catalytic reduction reaction is carried out in a buffer solution; and the buffer solution comprises a phosphate buffer, a Tris-HCl-based buffer, or a TEA-HCl-based buffer.

Preferably, the catalytic reduction reaction is carried out at a pH in the range of 6.0 to 8.0, such as pH 6.0, pH 6.2, pH 6.5, pH 6.8, pH 7.0, pH 7.2, pH 7.5, pH 7.8 or pH 8.0, and other specific pH values within this range may also be selected and are not described in detail herein.

In the present invention, the reduction process is carried out using a chiral metal catalyst, and the specific method includes:

in a hydrogen gas atmosphere, catalytically reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II using a chiral metal catalyst to thereby obtain (S)-nicotine.

Preferably, the chiral metal catalyst comprises a chiral iridium-based catalyst, a chiral ruthenium-based catalyst or a chiral rhodium-based catalyst, and more preferably a chiral iridium-based catalyst.

Preferably, a ligand is added to the catalytic reduction reaction.

The ligand is selected from any one of the ligands or a combination of at least two ligands in Table 1, preferably (R,R)-f-SpiroPhos and (S,S)-Ph-BPE.

Table 1

Preferably, the catalytic reduction reaction is carried out at a temperature in the range of 50°C to 100°C, such as 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, other specific temperatures within this range may also be selected, but they are not described in detail herein.

Preferably, during the catalytic reduction reaction, the pressure of hydrogen gas is maintained in a range of 1.0 MPa to 6.0 MPa, such as 1.0 MPa, 2.0 MPa, 3.0 MPa, 4.0 MPa, 5.0 MPa or 6.0 MPa, and other specific pressure values within this range may also be selected, but they are not described in detail herein.

Preferably, the catalytic reduction reaction is carried out at a pH in the range of 4.0 to 13.0, such as pH 4.0, 5.0, 6.0, pH 6.2, pH 6.5, pH 6.8, pH 7.0, pH 7.2, pH 7.5, pH 7.8, pH 8.0, pH 10.0, pH 12.0 or pH 13.0, and other specific pH values within this range may also be selected, but are not described in detail herein.

In the present invention, an alkene compound represented by formula I or an iminium cationic compound represented by formula II is obtained by desalting and/or cyclizing a compound represented by formula III or a salt thereof:

Preferably, the salt comprises hydrochloride, dihydrochloride, hydrobromide, dihydrobromide, sulfate or bisulfate.

The compound represented by formula III or a salt thereof is subjected to desalting and/or cyclization in the presence of an inorganic base and/or an organic base to thereby obtain an alkene compound represented by formula I or an iminium cationic compound represented by formula II.

In the present invention, a method for synthesizing a salt of a compound represented by formula III comprises:

mixing compound A with N-methylpyrrolidone and an organic base to obtain compound D, and mixing compound D with an acid to obtain a salt of the compound represented by formula III; the reaction equations are as follows:

.

Alternatively, a method for synthesizing a salt of a compound represented by formula III comprises:

mixing compound A with N-methylpyrrolidone and an organic base and neutralizing with an acid to achieve a pH of 7-8, thereby obtaining compound B, and mixing compound B with an acid to obtain a salt of a compound represented by formula III; the reaction equations are as follows:

.

In the present invention, a method for synthesizing compound A comprises: mixing nicotinic acid with methanol and esterifying in a highly acidic medium to thereby obtain compound A.

As a preferred technical solution according to the present invention, the method for synthesizing (S)-nicotine comprises:

mixing nicotinic acid with methanol and esterifying in a highly acidic medium to thereby obtain compound A; mixing compound A with N-methylpyrrolidone and an organic base to obtain compound D, optionally followed by neutralization with an acid to obtain compound B; mixing compound B or compound D with an acid to thereby obtain a salt of the compound represented by formula III; desalting and cyclizing the salt of the compound represented by formula III, and reducing the resulting product using a biocatalytic method or a chiral metal catalyst to obtain (S)-nicotine; and the reaction equations are as follows:

.

Compared with the prior art, the present invention has the following advantages. Considering that most of the previously disclosed methods for producing (S)-nicotine generally include a methylation step to obtain (S)-nicotine, or methylation is still necessary when using enzymatic catalysis, it indicates that the available methods for producing (S)-nicotine have limitations. The present invention creatively develops a completely new method for efficiently producing (S)-nicotine without methylation, in other words, reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II. The method has the advantages of simple operations, safety and reliability, high yield and high purity, thereby increasing the number of synthesis options available for producing (S)-nicotine.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 is a nuclear magnetic resonance spectrum of (S)-nicotine obtained in accordance with the present invention.

Fig. 2 is a nuclear magnetic resonance spectrum of the hydrochloride salt of compound III obtained according to the present invention.

Fig. 3 is a nuclear magnetic resonance spectrum of compound B obtained according to the present invention.

DETAILED DESCRIPTION

The technical solutions of the present invention are further described below using specific embodiments. It will be understood by those skilled in the art that the examples are intended only to provide a better understanding of the present invention and should not be considered as specific limitations of the present invention.

In the following Production Examples and Examples, the purity of compound A is determined using high performance liquid chromatography (HPLC), and the method for calculating the yield of compound A is as follows:

.

The purity of compound B is determined using high performance liquid chromatography (HPLC), and the method for calculating the yield of compound A is as follows:

.

The purity of the compound represented by formula III or a salt thereof is determined using high performance liquid chromatography (HPLC), and the method for calculating the yield of the compound represented by formula III or a salt thereof is as follows:

.

The purity and optical purity of (S)-nicotine are determined using high performance liquid chromatography (HPLC), and the method for calculating the yield of (S)-nicotine is as follows:

.

Example of getting 1-1

In this example of preparation, compound A was obtained:

.

50 g of nicotinic acid and 250 g of methanol were added to a 500 ml reaction vessel. With continuous stirring, 60 g of concentrated sulfuric acid was gradually added to the reaction vessel to form a mixture. The temperature was then raised and the mixture was heated under reflux for 18 hours and cooled to 45°C. The pressure was then reduced to concentrate the mixture, thereby removing methanol. After distilling off the residue, if any, the concentrated residue was cooled to 20°C. Then 250 g of ethyl acetate and 250 g of water were added and stirred until completely dissolved. The pH of the resulting mixture was adjusted to 7.5 using 20% sodium hydroxide solution and the resulting mixture was separated into an organic layer and an aqueous layer. The organic layer was collected. The aqueous layer was extracted once with 250 g of ethyl acetate, combined with the organic layer and dried with anhydrous sodium sulfate. The solvent was removed under reduced pressure to thereby obtain 55.2 g of a colorless transparent liquid with a purity of 99% and a yield of 99%. The liquid was cooled and allowed to solidify to obtain compound A.

Example of getting 1-2

In this preparation example, compound A was obtained.

50 g of nicotinic acid and 250 g of methanol were added to a 500 ml reaction vessel. Under continuous stirring, 120.5 g of thionyl chloride (2.5 equivalents) were added dropwise to the reaction vessel to form a mixture. The temperature was then raised and the mixture was heated under reflux for 9 hours and cooled to 40°C. The pressure was then reduced to concentrate the mixture, thereby removing methanol. After distilling off the residue, if any, the concentrated residue was cooled to 10°C. Then 200 g of dichloromethane and 100 g of water were added and stirred until completely dissolved. The pH of the resulting mixture was adjusted to 8 using 15% sodium hydroxide solution and the resulting mixture was separated into an organic layer and an aqueous layer. The organic layer was collected. The aqueous layer was extracted once with 200 g of dichloromethane, combined with the organic layer, washed with 5% sodium hydroxide solution and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to thereby obtain 44.8 g of a light yellow transparent liquid with a purity of 99% and a yield of 81%. The liquid was cooled and allowed to solidify to obtain compound A.

Example of getting 1-3

In this preparation example, compound A was obtained.

5 g of nicotinic acid, 5.2 g of trimethyl orthoformate (1.2 equivalents), 25 g of methanol and 0.48 g of zirconium tetrachloride (0.05 equivalents) were added to a 100 ml reaction vessel. Then, the temperature was raised with continuous stirring, and the mixture was heated under reflux for 17 hours and cooled to 25°C. Then, 0.55 g of sodium ethoxide (0.2 equivalents) was added to neutralize the cooled mixture, followed by filtration. The filtrate was concentrated under reduced pressure to remove methanol, thereby obtaining 5.5 g of a colorless transparent liquid with a purity of 98.1% and a yield of 99%. The liquid was cooled and allowed to solidify to obtain compound A.

Example of obtaining 1-4

In this preparation example, compound A was obtained. 5 g of nicotinic acid, 5.2 g of trimethyl orthoformate (1.2 equivalents), 25 g of methanol and 0.1 g of zirconium tetrachloride (0.01 equivalents) were added to a 100 ml reaction vessel. Then, the temperature was raised with continuous stirring, and the mixture was heated under reflux for 36 hours and cooled to 30°C. Then, 0.11 g of sodium ethoxide (0.04 equivalents) was added to neutralize the cooled mixture, followed by filtration. The filtrate was concentrated under reduced pressure to remove methanol, thereby obtaining 5.3 g of a colorless transparent liquid with a purity of 91.6% and a yield of 95%. The liquid was cooled and allowed to solidify, to obtain compound A.

Example of getting 1-5

In this preparation example, compound A was obtained.

5 g of nicotinic acid, 5.2 g of trimethyl orthoformate (1.2 equivalents), 25 g of methanol and 0.19 g of zirconium tetrachloride (0.02 equivalents) were added to a 100 ml reaction vessel. Then, the temperature was raised with continuous stirring, and the mixture was heated under reflux for 30 hours and cooled to 20°C. Then, 0.22 g of sodium ethoxide (0.08 equivalents) was added to neutralize the cooled mixture, followed by filtration. The filtrate was concentrated under reduced pressure to remove methanol, thereby obtaining 5.4 g of a colorless transparent liquid with a purity of 93.9% and a yield of 97%. The liquid was cooled and allowed to solidify to obtain compound A.

Example of getting 1-6

In this preparation example, compound A was obtained.

200 g of nicotinic acid, 207 g of trimethyl orthoformate (1.2 equivalents), 1000 g of methanol and 9.5 g of zirconium tetrachloride (0.025 equivalents) were added to a 2-L reaction vessel. Under continuous stirring, the temperature was raised and the mixture was heated under reflux for 20 hours and cooled to 25°C. Then, 11.06 g of sodium ethoxide (0.1 equivalents) was added to neutralize the cooled mixture, followed by filtration. The filtrate was concentrated under reduced pressure to remove methanol, thereby obtaining 220.6 g of a colorless transparent liquid with a purity of 98.5% and a yield of 99%. The liquid was cooled and allowed to solidify to obtain compound A.

Example of obtaining 2-1

In this example of preparation, compound B was obtained:

.

102.0 g of compound A obtained in Production Example 1-1, 88.5 g of N-methylpyrrolidone (1.2 equivalents), 1.2 kg of toluene and 133.6 g of potassium tert-butoxide (1.6 equivalents) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 25°C, neutralized to pH 7.5 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with dichloromethane, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, thereby obtaining 121.0 g of a yellow-brown liquid identified as compound B with a purity of 94% and a yield of 80%. No further purification steps were required, and compound B was directly fed to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H). The hydrogen NMR spectrum is shown in Fig. 3.

Example of getting 2-2

In this preparation example, compound B was obtained.

102.0 g of compound A obtained in Production Example 1-2, 77.4 g of N-methylpyrrolidone (1.05 equivalents), 1.2 kg of toluene and 91.8 g of potassium tert-butoxide (1.1 equivalents) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 20°C, neutralized to pH 7 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with toluene, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, to thereby obtain 118.0 g of a yellow-brown liquid identified as compound B with a purity of 93.5% and a yield of 78%. No further purification steps were required, and compound B was directly fed to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H).

Example of getting 2-3

In this preparation example, compound B was obtained.

102.0 g of compound A obtained in Production Example 1-3, 88.5 g of N-methylpyrrolidone (1.2 equivalents), 1.2 kg of toluene and 114.4 g of sodium tert-butoxide (1.6 equivalents) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 30°C and neutralized to pH 8 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with ethyl acetate, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, thereby obtaining 117.8 g of a yellow-brown liquid identified as compound B with a purity of 95% and a yield of 78%. No further purification steps were required, and compound B was directly fed to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H).

Example of obtaining 2-4

In this preparation example, compound B was obtained.

102.0 g of compound A obtained in Production Example 1-4, 77.4 g of N-methylpyrrolidone (1.05 equivalents), 1.2 kg of toluene and 78.6 g of sodium tert-butoxide (1.1 equivalents) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 25°C and neutralized to pH 7.5 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with toluene, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, thereby obtaining 108.7 g of a yellow-brown liquid identified as compound B with a purity of 93% and a yield of 72%. No further purification steps were required, and compound B was directly fed to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H).

Example of getting 2-5

In this preparation example, compound B was obtained.

102.0 g of compound A obtained in Production Example 1-5, 88.5 g of N-methylpyrrolidone (1.2 equivalents), 1.2 kg of toluene and 81.0 g of sodium ethoxide (1.6 equivalents) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating for 16 hours. After completion of the reaction, the mixture was cooled to 25°C and neutralized to pH 7.5 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with toluene, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, thereby obtaining 127.0 g of a yellow-brown liquid identified as compound B with a purity of 92% and a yield of 84%. No further purification steps were required, and compound B was directly fed to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H).

Example of obtaining 2-6

In this preparation example, compound B was obtained.

102.0 g of the compound A obtained in Production Example 1-6, 88.5 g of N-methylpyrrolidone (1.2 equivalents), 1.2 kg of toluene and 47.6 g of sodium hydride (1.6 equivalents, 60% sodium hydride in oil) were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 25°C and neutralized to pH 7 with 5% hydrochloric acid, followed by separation into an organic layer and an aqueous layer. The aqueous layer was extracted with methyl tert-butyl ether, combined with the organic layer, and concentrated under reduced pressure to remove the solvent, to thereby obtain 111.0 g of a yellow-brown liquid identified as compound B with a purity of 90% and a yield of 70%. No additional purification steps were required and compound B was directly carried on to the next reaction step.

The crude product was purified by column chromatography and analyzed using nuclear magnetic resonance (NMR). NMR analysis showed the simultaneous existence of both keto and enol forms, with the keto form predominating at a ratio of 10:1. The hydrogen NMR spectrum data for the keto form of the compound are presented as follows: δ ppm (400 MHz, CDCl ₃ ) 9.30 (s, 1H), 8.77-8.80 (m, 1H), 8.44 (1H, dt, J = 8.0, 2.0 Hz), 7.43-7.47 (m, 1H), 4.46 (1H, dd, J = 8.8, 3.2 Hz), 3.36-3.46 (m, 2H), 2.87 (s, 3H), 2.67-2.75 (m, 1H), 2.22-2.32 (m, 1H).

Example of obtaining 3-1

In this preparation example, the hydrochloride salt of the compound represented by formula III was prepared using compound B as a starting material,

100.0 g of the compound B obtained in Production Example 2-1 and 500.0 g of concentrated hydrochloric acid were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 55°C and concentrated under reduced pressure. 300.0 g of methanol was added to the resulting residue, cooled to 4°C, and subjected to filtration, thereby obtaining 92.2 g of an off-white solid identified as a compound represented by the formula III with a purity of 99% and a yield of 75%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the obtained compound corresponded to the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H). The spectrum is shown in Fig. 2.

Example of getting 3-2

In this preparation example, a compound represented by formula III was prepared using compound B as a starting material.

100.0 g of the compound B obtained in Production Example 2-2 and 100.0 g of concentrated hydrochloric acid were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 50°C and concentrated under reduced pressure. 100.0 g of methanol was added to the resulting residue, cooled to 8°C, and subjected to filtration, thereby obtaining 95.9 g of an off-white solid identified as a compound represented by the formula III with a purity of 98.5% and a yield of 78%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the obtained compound corresponded to the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of getting 3-3

In this preparation example, a compound represented by formula III was prepared using compound B as a starting material.

100.0 g of the compound B obtained in Production Example 2-3 and 300.0 g of concentrated hydrochloric acid were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 50°C and concentrated under reduced pressure. 300.0 g of ethanol was added to the resulting residue, cooled to 0°C, and subjected to filtration, thereby obtaining 100.8 g of an off-white solid identified as the compound represented by the formula III with a purity of 99% and a yield of 82%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the obtained compound corresponded to the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of getting 3-4

In this preparation example, a compound represented by formula III was prepared using compound B as a starting material.

100.0 g of the compound B obtained in Production Example 2-4 and 300.0 g of concentrated hydrochloric acid were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 55°C and concentrated under reduced pressure. 500.0 g of isopropanol was added to the resulting residue, cooled to 4°C, and subjected to filtration, thereby obtaining 93.4 g of an off-white solid identified as the compound represented by the formula III with a purity of 97.5% and a yield of 76%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the obtained compound corresponded to the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of getting 3-5

In this preparation example, a compound represented by formula III was prepared using compound B as a starting material.

100.0 g of the compound B obtained in Production Example 2-5 and 500.0 g of concentrated hydrochloric acid were added to a reaction vessel. Under continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 55°C and concentrated under reduced pressure. 100.0 g of isopropanol was added to the resulting residue, cooled to 4°C, and subjected to filtration, thereby obtaining 103.3 g of an off-white solid identified as the compound represented by the formula III with a purity of 96% and a yield of 84%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the obtained compound corresponded to the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of obtaining 4-1

In this preparation example, compound D was first prepared from compound A as a starting material, followed by obtaining a compound represented by formula III.

120.0 g of compound A obtained in Production Example 1-1, 96.2 g of N-methylpyrrolidone, 1.6 kg of toluene and 147 g of potassium tert-butoxide were added to a reaction vessel. While continuously stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 25°C and filtered to thereby obtain compound D (potassium salt). Compound D was directly supplied to the next reaction step.

Compound D (potassium salt) obtained by filtration was added to 650 g of concentrated hydrochloric acid, and the temperature was raised to 100°C. After completion of the reaction, distillation under reduced pressure was employed to remove most of the water from the resulting mixture. Then, 650 ml of 95% ethanol was added to the residue, followed by thorough stirring. The temperature was lowered to 10°C, and the mixture was subjected to filtration and drying, thereby obtaining 86 g of an off-white solid identified as the compound represented by formula III, in a yield of 55.2% and a purity of 97.2%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the compound represented by formula III was consistent with the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of getting 4-2

In this preparation example, compound D was first prepared from compound A as a starting material, followed by obtaining a compound represented by formula III.

153.0 g of compound A obtained in Production Example 1-2, 165.0 g of N-methylpyrrolidone, 2 kg of toluene and 214.5 g of potassium tert-butoxide were added to a reaction vessel. With continuous stirring, the temperature was raised and the mixture was subjected to reflux heating. After completion of the reaction, the mixture was cooled to 15°C and filtered to thereby obtain compound D (potassium salt). Compound D was directly supplied to the next reaction step.

Compound D (potassium salt) obtained by filtration was added to 960 g of concentrated hydrochloric acid, and the temperature was raised to 100°C. After completion of the reaction, distillation under reduced pressure was employed to remove most of the water from the resulting mixture. Then, 800 ml of isopropanol was added to the residue, followed by thorough stirring. The temperature was lowered to 20°C, and the mixture was subjected to filtration and dried, thereby obtaining 127 g of an off-white solid identified as the compound represented by formula III, with a yield of 63.5% and a purity of 96.7%.

When analyzed, the nuclear magnetic resonance spectrum ( ¹ H-NMR) of the compound represented by formula III was consistent with the predicted structure, and the spectral data were as follows: ¹ H-NMR (400 MHz, D ₂ O): δ ppm 9.25 (m, 1H), 9.0 (dt, J = 8.4 Hz, 1.6 Hz, 1H), 8.90-8.91 (m, 1H), 8.13-8.17 (m, 1H), 3.28 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 2.66 (s, 3H), 2.02-2.09 (m, 2H).

Example of getting 5-1

In this production example, glucose dehydrogenase with the amino acid sequence presented under SEQ ID No: 1 was obtained.

The amino acid sequence of glucose dehydrogenase (SEQ ID No: 1) obtained from Priestia megaterium (NCBI accession number AUO12718.1) was sent to Kingsray Corporation in Nanjing for codon optimization and total gene synthesis. Subsequently, the amino acid sequence was ligated into pET30a(+) plasmid and transformed into Escherichia coli BL21 (DE3) competent cells, thereby obtaining recombinant bacteria containing the glucose dehydrogenase gene.

The recombinant bacteria were inoculated into 5 ml of LB liquid medium containing 50 μg/ml kanamycin and cultured overnight at 37°C. Then, 1 ml of the bacterial culture was transferred to 125 ml of LB liquid medium containing 50 μg/ml kanamycin and incubated at 37°C for 3 h; subsequently, 125 μl of 1 M IPTG were added and the culture was induced overnight at 25°C. The cells were collected by centrifugation (4000 rpm and 4°C for 10 min), resuspended in phosphate buffer (pH 7.0) at four times the original volume, sonicated to ensure cell disruption, and then centrifuged (4000 rpm and 4°C for 10 min) to collect the supernatant. The supernatant was lyophilized to obtain a powder of the glucose dehydrogenase enzyme.

Example of getting 5-2

In this preparation example, the following eleven different imine reductases were prepared, designated as enzymes 1, 3-11, and 13.

The amino acid sequences of imine reductases submitted to NCBI (with information presented in the table below under SEQ ID No: 2-12) were sent to Kingsray Corporation in Nanjing for codon optimization and total gene synthesis. Subsequently, the amino acid sequences were ligated into pET30a(+) plasmid and transformed into competent Escherichia coli BL21 (DE3) cells, thereby obtaining recombinant bacteria containing the imine reductase gene.

The recombinant bacteria were inoculated into 5 ml of LB liquid medium containing 50 μg/ml kanamycin and cultured overnight at 37°C. Subsequently, 1 ml of the bacterial culture was transferred to 125 ml of LB liquid medium containing 50 μg/ml kanamycin and incubated at 37°C for 3 h; then 125 μl of 1 M IPTG was added and the culture was induced for 16 h at 25°C. The cells were collected by centrifugation (4000 rpm, 4°C for 10 min), resuspended in phosphate buffer (pH 7.0) at four times the original volume, sonicated to ensure cell disruption, and then centrifuged again (4000 rpm, 4°C for 10 min) to collect the supernatant. The resulting supernatant was lyophilized to obtain imine reductase powder.

Ten imine reductases designated as enzymes 14 to 23 were also obtained in this production example by performing site-directed mutagenesis on the amino acid sequence shown as SEQ ID NO: 12, thereby obtaining imine reductases containing the amino acid sequences shown as SEQ ID NO: 13 to 22. The amino acid sequences were sent to Kingsray Corporation in Nanjing for codon optimization and total gene synthesis. Subsequently, the amino acid sequences were ligated into the pET30a(+) plasmid and transferred into Escherichia coli BL21 (DE3) competent cells, thereby obtaining recombinant bacteria containing the imine reductase genes. The same operations were carried out to finally obtain the imine reductase powder.

Example of getting 5-3

In this preparation example, the following enzyme was obtained, designated as enzyme 12.

The amino acid sequence (SEQ ID No: 2) of imine reductase and the amino acid sequence (SEQ ID No: 1) of glucose dehydrogenase were sequentially cloned into the plasmid pETDuet-1. The recombinant plasmid was then transformed into competent Escherichia coli BL21 (DE3) cells, thereby obtaining recombinant bacteria containing both the imine reductase gene and the glucose dehydrogenase gene.

The recombinant bacteria were inoculated into 5 ml of LB liquid medium containing 100 μg/ml ampicillin and cultured overnight at 37°C. Subsequently, 1 ml of the bacterial culture was transferred to 125 ml of LB liquid medium containing 100 μg/ml ampicillin and incubated at 37°C for 3 hours; then 125 μl of 1 M IPTG was added and the culture was induced overnight at 25°C. The cells were collected by centrifugation (4000 rpm, 4°C for 10 min), resuspended in phosphate buffer (pH = 7.0) at four times the original volume, sonicated to ensure cell disruption, and then centrifuged again (4000 rpm, 4°C for 10 min) to collect the supernatant. The resulting supernatant was lyophilized to obtain enzyme powder 12.

Example 1

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of compound C obtained in Production Example 3-1, 50 ml of methanol, 1.0 g of triethylamine, 16.7 mg of chloro(1,5-cyclooctadiene)iridium(I) dimer as a chiral metal catalyst, and 30 mg of (S)-(-)-1-((R)-2-diphenylphosphino)ferrocenyl)ethyl di-tert-butylphosphine as a ligand were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain a pressure of 2.5 MPa, and the temperature was maintained at 60°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in n-heptane, filtered, and concentrated to obtain (S)-nicotine with a purity of 96% and an optical purity of 72%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR), as shown in Fig. 1. The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 2

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of compound C obtained in Production Example 3-2, 50 ml of methanol, 1.0 g of triethylamine, 16.7 mg of chloro(1,5-cyclooctadiene)iridium(I) dimer as a chiral metal catalyst, and 27 mg of (+)-1,2-bis((2S,5S)-2,5-diphenylphospholano)ethane as a ligand were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain the pressure at 3.0 MPa, and the temperature was maintained at 60°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in n-heptane, filtered, and concentrated to obtain (S)-nicotine with a purity of 94% and an optical purity of 80%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 3

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of compound C obtained in Production Example 3-3, 50 ml of methanol, 1.0 g of triethylamine, 16.7 mg of chloro(1,5-cyclooctadiene)iridium(I) dimer as a chiral metal catalyst, and 38 mg of (R,R)-f-SpiroPhos as a ligand were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain the pressure at 5.5 MPa, and the temperature was maintained at 80°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in n-heptane, filtered, and concentrated to obtain (S)-nicotine with a purity of 90% and an optical purity of 88%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 4

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

Example 4 is similar to Example 3 except for the following differences: 16.7 mg of chloro(1,5-cyclooctadiene)iridium(I) dimer and 38 mg of R,R)-f-SpiroPhos were replaced by 45 mg of Rh((R,R)-DIPAMP)(COD) _BF4 , while all other conditions were left unchanged. The resulting (S)-nicotine exhibited a purity of 45% and an optical purity of 27%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 5

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of the compound C obtained in Production Example 3-4, 60 ml of ethanol, 1.0 g of triethylamine and 45 mg of Rh[(R,R)-DIPAMP](COD) _BF4 as a chiral metal catalyst were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain the pressure at 4.0 MPa and the temperature was maintained at 90°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in n-heptane, filtered and concentrated to obtain (S)-nicotine with a purity of 85% and an optical purity of 58%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 6

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of the compound C obtained in Production Example 3-5, 50 ml of methanol, 1.0 g of N,N-diisopropylethylamine and 39 mg of (-)-1,2-bis((2S,5S)-2,5-dimethylphosphino)ethane(cyclooctadiene)rhodium(I) tetrafluoroborate as a chiral metal catalyst were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain the pressure at 1.5 MPa and the temperature was maintained at 100°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in methyl tert-butyl ether, filtered and concentrated to obtain (S)-nicotine with a purity of 64% and an optical purity of 82%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 7

In this example, (S)-nicotine was prepared by a reduction process using a chiral metal catalyst.

1.25 g of compound C obtained in Production Example 4-1, 50 ml of methanol, 1.0 g of triethylamine and 35 mg of [(R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]dichlororuthenium(II) as a chiral metal catalyst were added to a 100 ml hydrogenation reactor. Then, hydrogen gas was introduced into the hydrogenation reactor to maintain the pressure at 3.0 MPa and the temperature was maintained at 90°C. When the reaction was completed, the solvent was removed by concentration, and the residue was suspended in n-heptane, filtered and concentrated to obtain (S)-nicotine with a purity of 59% and an optical purity of 66%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 8

In this example, (S)-nicotine was obtained using a biocatalytic method.

30 mg of Compound C obtained in Production Example 4-2 were added to each of 10 centrifuge tubes (each 5 ml), followed by adding 2 ml of 0.1 M phosphate buffer. The pH of the mixture was adjusted to 6.0. Then, 32 mg of glucose was added to each centrifuge tube and stirred until completely dissolved. Subsequently, 30 mg of the imine reductases containing the amino acid sequences shown as SEQ ID NOS: 2-22 obtained in Production Examples 5-2 were separately added to each centrifuge tube. 3 ml of 0.1 M phosphate buffer, 40 mg of enzyme 2 obtained in Production Example 5-1 and 40 mg of NADP salt were added to the second 5 ml centrifuge tube and stirred until completely dissolved. Then 0.1 ml of the solution from the second centrifuge tube was slowly added to each of the first centrifuge tubes. The temperature was raised to 25°C and the mixture was stirred at 300 rpm for 16 hours. 0.1 ml of the resulting solution was mixed with 0.9 ml of methanol, shaken for 1 min, filtered and then transferred to a 1 ml liquid phase vial for analysis using high performance liquid chromatography. The area ratio of (S)-nicotine corresponds to the degree of conversion.

The conversion data are shown in Table 2.

Table 2

Referring to the data in Table 2, it is evident that enzymes 1, 3, 4, 13-18, and 22 exhibit superior catalytic efficiency compared with other enzymes, achieving a conversion rate of 94.6% or more.

Example 9

In this example, (S)-nicotine was obtained using a biocatalytic method.

4.5 g of Compound C obtained in Preparation Example 4-1 and 20 ml of 0.1 M phosphate buffer were added to a 50 ml three-necked round-bottomed flask, and the pH of the mixture was adjusted to 7.0. Then, 4.8 g of glucose was added to the three-necked round-bottomed flask and stirred until completely dissolved. 10 ml of 0.1 M phosphate buffer, 0.3 g of Enzyme 1 obtained in Preparation Example 5-2, 0.04 g of Enzyme 2 obtained in Preparation Example 5-1, and 0.008 g of NADP salt were added to a 50 ml flask and stirred until completely dissolved. Then, the solution in the flask was slowly added to the three-necked round-bottomed flask. The temperature was raised to 30°C, and the resulting mixture was stirred at 300 rpm for 16 hours and filtered. The pH of the filtrate was adjusted to 10 with sodium hydroxide solution. The filtrate was then extracted with methyl tert-butyl ether, dried with anhydrous sodium sulfate, and concentrated to obtain 2.6 g of (S)-nicotine with a purity of 99%, an optical purity of 100%, and a yield of 89.5%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 10

In this example, (S)-nicotine was obtained using a biocatalytic method.

4.5 g of compound C obtained in Production Example 3-1 and 20 ml of 0.1 M phosphate buffer were added to a 50 ml three-necked round bottom flask, and the pH of the mixture was adjusted to 6.0. Then, 4.8 g of glucose was added to the three-necked round bottom flask and stirred until completely dissolved. 10 ml of 0.1 M phosphate buffer, 0.4 g of enzyme 3 obtained in Production Example 5-2, 0.04 g of enzyme 2 obtained in Production Example 5-1, and 0.008 g of NADP salt were added to a 50 ml flask and stirred until completely dissolved. Then, the solution in the flask was slowly added to the three-necked round bottom flask. The temperature was raised to 35°C, and the resulting mixture was stirred at 300 rpm for 16 hours and filtered. The pH of the filtrate was adjusted to 10 with sodium hydroxide solution. The filtrate was then extracted with ethyl acetate, dried with anhydrous sodium sulfate, and concentrated to obtain 2.4 g of (S)-nicotine with a purity of 99%, an optical purity of 99.4%, and a yield of 82.6%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

Example 11

In this example, (S)-nicotine was obtained using a biocatalytic method.

4.5 g of compound C obtained in Preparation Example 3-1 and 20 ml of 0.1 M phosphate buffer were added to a 50 ml three-neck round-bottomed flask, and the pH of the mixture was adjusted to 6.0. Then, 4.8 g of glucose was added to the three-neck round-bottomed flask and stirred until completely dissolved. 10 ml of 0.1 M phosphate buffer, 0.4 g of enzyme 12 obtained in Preparation Example 5-3, and 0.008 g of NADP salt were added to the 50 ml flask and stirred until completely dissolved. Then, the solution in the flask was slowly added to the three-neck round-bottomed flask. The temperature was raised to 35°C, and the resulting mixture was stirred at 300 rpm for 16 hours and filtered. The pH of the filtrate was adjusted to 10 with sodium hydroxide solution. The filtrate was then extracted with ethyl acetate, dried with anhydrous sodium sulfate, and concentrated to obtain 2.3 g of (S)-nicotine with a purity of 99%, an optical purity of 99.6%, and a yield of 79.2%.

The obtained (S)-nicotine was purified by column chromatography and characterized using nuclear magnetic resonance (NMR). The NMR data are as follows: ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.54 (1H, d), 8.50 (1H, dd), 7.70 (1H, dt), 7.24-7.27 (1H, m), 3.22-3.27 (1H, m), 3.08 (1H, t), 2.27-2.34 (1H, m), 2.17-2.24 (1H, m), 2.16 (3H, m), 1.91-2.02 (1H, m), 1.79-1.87 (1H, m), 1.68-1.76 (1H, m). The results confirm the successful synthesis of (S)-nicotine.

The applicant states that the present invention illustrates a method for producing (S)-nicotine using the recovery method of the above embodiments, but the present invention is not limited to the above embodiments, which does not mean that it is necessary to rely on the above embodiments to carry out the present invention. It should be understood by those skilled in the art that any improvements of the present invention, equivalent replacement of starting materials and addition of auxiliary components to the product of the present invention, as well as the choice of specific methods are within the scope of legal protection and disclosure of the present invention.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, many simple changes can be made to the technical solution of the present invention, all of which are within the scope of legal protection of the present invention.

In addition, it should be noted that the specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, all possible combined methods are not described in the present invention.

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SEQUENCE LIST

<110> PORTON PHARMA SOLUTIONS LTD.

<120> METHOD OF USING REDUCTION TO OBTAIN (S)-NICOTINE

<130>BY22DX0700FPPC-CN

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 260

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Glucose dehydrogenase

<400> 1

Met Tyr Lys Asp Leu Glu Gly Lys Val Val Val Ile Thr Gly Ser Ser

1 5 10 15

Thr Gly Leu Gly Lys Ser Met Ala Ile Arg Phe Ala Thr Glu Lys Ala

20 25 30

Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val

35 40 45

Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly

50 55 60

Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile

65 70 75 80

Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu

85 90 95

Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Asn Lys Val

100 105 110

Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg

260

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Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Glu Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ser Leu Glu Ser Trp Cys His Thr Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Asp Gln Ile Gly Thr Thr Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Thr Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Pro Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Val Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

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Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 4

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<223> Imin reductase

<400> 4

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Ile Lys Gly Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Arg Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Asp Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Tyr Phe Ala Gly Gly

130 135 140

Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu Gly Leu

145 150 155 160

Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro Ile Leu

165 170 175

Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly Asp Phe

180 185 190

Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile Ser Arg

195 200 205

Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe Pro Arg

210 215 220

Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe Gly Gln

225 230 235 240

Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn Gly Ala

245 250 255

Ala Leu

<210> 5

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<223> Imin reductase

<400> 5

Met Arg Pro Ile Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Thr Pro Leu Ile Ala Leu Gly Ala Ile Leu Ala

35 40 45

Pro Ser Val Ser Glu Ala Ile Ala Ala Gly Asp Ile Thr Leu Ile Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asn

65 70 75 80

Ala Val Thr Gly Lys Leu Val Val Gln Leu Ser Thr Gly Ser Pro Leu

85 90 95

Gly Ala Arg Thr Leu Glu Ser Trp Cys His Ala Arg Gly Ala Cys Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Asp Gln Ile Gly Thr Thr Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Asn Ala Ala Phe Arg Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Glu His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Thr Asp His Ala Ala Asp Ala Gly Ile Asp Asn Ser Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Glu Gln Gly Leu

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 6

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<223> Imin reductase

<400> 6

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Ile Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ser Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Gln Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Asp Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Asn Thr Ala Phe Ser Asp Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Thr Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 7

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<223> Imin reductase

<400> 7

Met Gly Thr Ala Leu Val Glu Ala Phe Leu Ala Gly Gly His Ala Thr

1 5 10 15

Thr Val Trp Asn Arg Thr Pro Gly Lys Ala Asp Gly Val Val Ala Arg

20 25 30

Gly Ala Val Val Ala Glu Thr Val Ala Glu Ala Val Ala Ala Ser Pro

35 40 45

Leu Val Val Val Cys Leu Trp Asp Asp Ala Val Val Arg Asp Val Leu

50 55 60

His Pro Val Ala Asp Ala Leu Ala Gly Arg Val Val Val Asn Leu Thr

65 70 75 80

Asn Gly Thr Pro Ala Gln Ala Arg Glu Met Ala Ala Trp Ala Ala Glu

85 90 95

His Gly Val Glu Tyr Val Asp Gly Gly Ile Met Ala Ile Pro Pro Gly

100 105 110

Ile Gly Thr Glu His Ala Phe Val Leu Tyr Ser Gly Ala Glu Ala Ala

115 120 125

Phe Glu Ala His Arg Glu Val Leu Glu Arg Leu Gly Ala Ala Lys Tyr

130 135 140

Leu Gly Ala Asp Ala Gly Leu Ala Ala Leu Phe Asp Leu Ala Leu Leu

145 150 155 160

Ser Gly Met Tyr Gly Thr Phe Ala Gly Leu Trp His Ser Leu Ala Met

165 170 175

Val Arg Thr Glu Asn Val Ser Ala Ala Glu Phe Val Pro Met Leu Gly

180 185 190

Pro Trp Met Gln Ala Met Ile Gly Gly Asn Leu Asp Arg Leu Ala His

195 200 205

Gln Leu Asp Thr Gly Asp Tyr Gly His Glu Val Val Ser Asn Leu Ala

210 215 220

Met Gln Ala Ala Ala Phe Pro Asn Ile Val Gln Ala Ser Leu Asp Gln

225 230 235 240

Gly Ile Arg Pro Asp Leu Met Ala Pro Ile Gln Arg Leu Met Asp Gln

245 250 255

Ala Val Ala Ala Gly His Gly Ala Glu Asp Val Ala Val Val Val Asp

260 265 270

Leu Leu Lys Asn

275

<210> 8

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<223> Imin reductase

<400> 8

Met Lys Pro Thr Leu Thr Val Ile Gly Ala Gly Arg Met Gly Ser Ala

1 5 10 15

Leu Ile Lys Ala Phe Leu Gln Ser Gly Tyr Thr Thr Thr Val Trp Asn

20 25 30

Arg Thr Lys Ala Lys Ser Glu Pro Leu Ala Lys Leu Gly Ala His Leu

35 40 45

Ala Asp Thr Val Arg Asp Ala Val Lys Arg Ser Asp Ile Ile Val Val

50 55 60

Asn Val Leu Asp Tyr Asp Thr Ser Asp Gln Leu Leu Arg Gln Asp Glu

65 70 75 80

Val Thr Arg Glu Leu Arg Gly Lys Leu Leu Val Gln Leu Thr Ser Gly

85 90 95

Ser Pro Ala Leu Ala Arg Glu Gln Glu Thr Trp Ala Arg Gln His Gly

100 105 110

Ile Asp Tyr Leu Asp Gly Ala Ile Met Ala Thr Pro Asp Phe Ile Gly

115 120 125

Gln Ala Glu Cys Ala Leu Leu Tyr Ser Gly Ser Ala Ala Leu Phe Glu

130 135 140

Lys His Arg Ala Val Leu Asn Val Leu Gly Gly Ala Thr Ser His Val

145 150 155 160

Gly Glu Asp Val Gly His Ala Ser Ala Leu Asp Ser Ala Leu Leu Phe

165 170 175

Gln Met Trp Gly Thr Leu Phe Gly Thr Leu Gln Ala Leu Ala Ile Ser

180 185 190

Arg Ala Glu Gly Ile Pro Leu Glu Lys Thr Thr Ala Phe Ile Lys Leu

195 200 205

Thr Glu Pro Val Thr Gln Gly Ala Val Ala Asp Val Leu Thr Arg Val

210 215 220

Gln Gln Asn Arg Leu Thr Ala Asp Ala Gln Thr Leu Ala Ser Leu Glu

225 230 235 240

Ala His Asn Val Ala Phe Gln His Leu Leu Ala Leu Cys Glu Glu Arg

245 250 255

Asn Ile His Arg Gly Val Ala Asp Ala Met Tyr Ser Val Ile Arg Glu

260 265 270

Ala Val Lys Ala Gly His Gly Lys Asp Asp Phe Ala Ile Leu Thr Arg

275 280 285

Phe Leu Lys

290

<210> 9

<211> 329

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 9

Met Val Ser Ser Pro Tyr Leu Asn Val Thr Ala Tyr Pro Lys Val Arg

1 5 10 15

Asn Leu Pro Trp Pro Val Pro Gly Pro Ile Arg Val Ala Ser Gln Ile

20 25 30

Leu Glu Leu Arg Pro Met Thr Thr Ile Gly Phe Leu Gly Ala Gly Arg

35 40 45

Met Gly Ser Ala Leu Val Lys Ser Leu Leu Glu Ala Gly His Ser Val

50 55 60

His Val Trp Asn Arg Thr Ala Glu Lys Ala Gln Ala Leu Ala Asp Phe

65 70 75 80

Gly Ala Val Pro Glu Pro Ser Ala Glu Arg Ala Ala Gly Pro Ala Glu

85 90 95

Ile Val Ile Val Asn Leu Leu Asp Tyr Glu Ala Ser Asp Ala Glu Leu

100 105 110

Arg Lys Pro Asp Val Ala Glu Ala Leu Lys Gly Lys Leu Leu Val Gln

115 120 125

Leu Thr Ser Gly Ser Pro Lys Thr Ala Arg Glu Thr Gly Arg Trp Ala

130 135 140

Gly Asp His Gly Ile Ala Tyr Leu Asp Gly Ala Ile Met Ala Thr Pro

145 150 155 160

Asn Phe Ile Gly Gly Ala Glu Thr Val Ile Leu Tyr Ser Gly Ser Lys

165 170 175

Thr His Phe Glu Lys His Glu Gly Leu Phe Lys Ala Leu Gly Gly Lys

180 185 190

Ser Ala Phe Val Gly Glu Asp Phe Gly Thr Ala Ser Ala Leu Asp Ser

195 200 205

Ala Leu Leu Ser Gln Met Trp Gly Thr Leu Phe Gly Thr Leu Gln Ala

210 215 220

Leu Ala Val Cys Arg Ala Glu Gly Ile Glu His Asp Val Tyr Ala Gly

225 230 235 240

Phe Leu Met Ser Ala Gln Pro Met Ile Asp Gly Ala Gln Gln Asp Leu

245 250 255

Met Glu Arg Ile Arg Asp Gly Arg Asp Leu Ala Asp Ala Gln Thr Leu

260 265 270

Ala Thr Val Ala Val His Asn Val Ala Phe His His Leu Arg Asp Leu

275 280 285

Ile Ala Asp Arg Asp Leu Asn Pro Ala Phe Gly Asp Ala Leu Gly Ser

290 295 300

Leu Leu Glu Thr Ala Leu Arg Asn Asp His Gln Asp Asp Asp Phe Ala

305 310 315 320

Val Leu Ala Arg Phe Met Gly Ala Lys

325

<210> 10

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<400> 10

Met Thr Asp Leu Gly Lys Ser Ala Val Thr Val Leu Gly Leu Gly Ala

1 5 10 15

Met Gly Thr Ala Leu Ala Glu Ala Leu Leu Ala Ala Gly His Pro Thr

20 25 30

Thr Val Trp Asn Arg Ser Pro Ala Arg Thr Ala Gly Pro Ala Gln Arg

35 40 45

Gly Ala Ala Val Ala Ala Ala Thr Ala Glu Ala Ile Ala Ala Ser Arg

50 55 60

Leu Ile Val Val Cys Leu Leu Asp His Thr Ser Val His Ala Val Leu

65 70 75 80

Asp Gly Gln Glu Leu Thr Gly Arg Ile Val Val Asn Leu Thr Ser Gly

85 90 95

Thr Pro Gly Gln Ala Arg Glu Leu Asp Ala Arg Val Ala Glu Arg Gly

100 105 110

Gly Asp His Leu Asp Gly Ala Val Leu Ala Val Pro Ser Met Ile Gly

115 120 125

Thr Pro Asp Ala Ser Val Leu Tyr Ser Gly Ser Arg Gly Ala Phe Asp

130 135 140

Thr His Arg Pro Val Leu Glu Val Phe Gly Ala Ala Asp Tyr Val Gly

145 150 155 160

Ala Asp Pro Gly Ala Ala Ser Leu Gln Asp Ala Ala Leu Leu Ser Ala

165 170 175

Met Tyr Gly Gln Val Ala Gly Val Leu His Ala Phe Ala Leu Val Arg

180 185 190

Ser Ala Gly Val Thr Ala Thr Glu Phe Leu Pro Arg Leu Val Gly Trp

195 200 205

Leu Thr Ala Met Gly Gly Phe Pro Ala Asp Ala Ala Arg Arg Ile Asp

210 215 220

Ala Arg Ala Tyr Ala Asp Asp Val Asp Ala Ala Leu Thr Met Gln Val

225 230 235 240

Thr Ala Val Arg Asn Leu Val Arg Ala Ala Arg Glu Gln Gly Val Ser

245 250 255

Ala Glu Leu Ile Ala Pro Leu Val Pro Val Met Gln Arg Arg Ile Asp

260 265 270

Asp Gly Asp Gly Gly Asp Asp Leu Ala Ala Leu Val Glu Val Ile Thr

275 280 285

Ala Glu Glu Val Ala

290

<210> 11

<211> 290

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 11

Met Thr Asp Lys Pro Pro Val Thr Val Leu Gly Leu Gly Ala Met Gly

1 5 10 15

Thr Ala Leu Ala Arg Thr Leu Leu Asn Ala Gly Tyr Pro Thr Thr Val

20 25 30

Trp Asn Arg Thr Ala Ser Lys Thr Ala Pro Leu Thr Glu Leu Gly Ala

35 40 45

His Ala Ala Asp Ser Pro Ala Asp Ala Ile Ala Arg Gly Glu Leu Val

50 55 60

Leu Ala Cys Leu Leu Asp Tyr Asp Ser Val His Gln Thr Leu Ala Gly

65 70 75 80

Thr Gly Asp Ala Leu Arg Gly Lys Ala Phe Val Asn Leu Thr Asn Gly

85 90 95

Thr Pro Glu Gln Ala Arg Ala Leu Ala Gly Lys Leu Asp Thr Ala Tyr

100 105 110

Leu Asp Gly Gly Ile Met Ala Val Pro Pro Met Ile Gly Ser Pro Gly

115 120 125

Ala Phe Leu Phe Tyr Ser Gly Glu Ile Ala Val Phe Glu Gln Tyr Arg

130 135 140

Pro Val Leu Glu Ser Phe Gly Glu Ala Ile Glu Val Gly Thr Asp Pro

145 150 155 160

Gly Leu Ala Ala Leu His Asp Leu Ala Leu Leu Ser Ala Met Tyr Gly

165 170 175

Met Phe Gly Gly Val Leu Gln Ala Phe Ala Leu Thr Gly Ser Ala Gly

180 185 190

Val Ser Ala Ala Ser Leu Ala Pro Leu Leu His Arg Trp Leu Asp Gly

195 200 205

Met Ser Gly Phe Ile Ala Gln Ser Ala Ala Gln Leu Asp Ser Gly Asp

210 215 220

Phe Ala Thr Gly Val Val Ser Asn Leu Ala Met Gln Asp Thr Gly Phe

225 230 235 240

Ala Asn Leu Phe Arg Ala Ala Lys Glu Gln Gly Ile Ser Thr Gly Gln

245 250 255

Leu Glu Pro Leu Gly Ala Leu Ile Arg Arg Arg Val Glu Asp Gly His

260 265 270

Gly Ala Glu Asp Leu Ala Gly Ile Val Glu Tyr Leu Lys Ile Gly Ala

275 280 285

Asn Ala

290

<210> 12

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 12

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 13

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 13

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Val Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Ala Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 14

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 14

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 15

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 15

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Ala Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Ala

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Gly Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 16

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 16

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Ala Val Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Thr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Gly Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 17

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 17

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Ala Val Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Gly Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 18

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 18

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Ala Ala Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Gly Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 19

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 19

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Leu Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Ser Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Val Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Gly Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 20

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 20

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 21

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 21

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Tyr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ala Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<210> 22

<211> 292

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Imin reductase

<400> 22

Met Arg His Leu Ser Val Ile Gly Leu Gly Ala Met Gly Ser Ala Leu

1 5 10 15

Ala Thr Thr Leu Leu Lys Ala Gly His Pro Val Thr Val Trp Asn Arg

20 25 30

Ser Ala Ala Lys Ala Ala Pro Leu Gln Ala Leu Gly Ala Thr Leu Ala

35 40 45

Pro Ser Val Gly Ala Ala Ile Ala Arg Ser Asp Ile Thr Leu Val Cys

50 55 60

Val Asp Asn Tyr Ala Val Ser Gln Gln Leu Leu Asp Glu Ala Ser Asp

65 70 75 80

Ala Val Ala Gly Lys Leu Leu Val Gln Leu Ser Thr Gly Ser Pro Gln

85 90 95

Gly Ala Arg Ala Leu Glu Ser Trp Ser His Ala Arg Gly Ala Arg Tyr

100 105 110

Leu Asp Gly Ala Ile Leu Cys Phe Pro Ala Gln Ile Gly Thr Ser Asp

115 120 125

Ala Ser Ile Ile Cys Ser Gly Ala Ser Ala Ala Phe Ser Glu Ala Glu

130 135 140

Pro Val Leu Arg Leu Leu Ala Pro Thr Leu Asp His Val Ala Glu Ala

145 150 155 160

Val Gly Ala Ala Ala Ala Gln Asp Cys Ala Val Ala Ala Tyr Phe Gly

165 170 175

Gly Gly Leu Leu Gly Ala Leu His Gly Ala Leu Ile Cys Glu Ala Glu

180 185 190

Gly Leu Pro Val Ala Lys Val Cys Ala Gln Phe Ser Glu Leu Ser Pro

195 200 205

Ile Leu Gly Gly Asp Val Ala His Leu Gly Lys Thr Leu Ala Ser Gly

210 215 220

Asp Phe Asp His Pro Thr Ala Ser Leu Lys Thr Trp Ser Ala Ala Ile

225 230 235 240

Ser Arg Leu Ala Gly His Ala Thr Asp Ala Gly Ile Asp Ser Arg Phe

245 250 255

Pro Arg Phe Ala Ala Asp Leu Phe Glu Glu Gly Val Ala Gln Gly Phe

260 265 270

Gly Gln Gln Glu Val Ser Ala Leu Ile Lys Val Leu Arg Ala Arg Asn

275 280 285

Gly Ala Ala Gln

290

<---

Claims (34)

1. A method for producing (S)-nicotine by reduction, comprising: carrying out a process of reducing an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II, thereby obtaining (S)-nicotine: Formula I; Formula II; in this case, the recovery process is carried out using a biocatalytic method and the biocatalytic method includes: a catalytic reduction of an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II, carried out in a coenzyme-cycling system using imine reductase as a catalyst for a catalytic reduction reaction to thereby obtain (S)-nicotine; wherein the coenzyme cycling system comprises a coenzyme, glucose and glucose dehydrogenase; and wherein the imine reductase comprises an amino acid sequence as set forth in SEQ ID No: 2-6 and SEQ ID No: 12-22; or The reduction process is carried out using a chiral metal catalyst and involves: catalytic reduction of an alkene compound represented by formula I and/or an iminium cationic compound represented by formula II, carried out in a hydrogen gas atmosphere, using a chiral metal catalyst to thereby obtain (S)-nicotine; wherein the chiral metal catalyst is selected from chloro(1,5-cyclooctadiene)iridium (I) dimer, (-)-1,2-bis((2S,5S)-2,5-dimethylphosphino)ethane(cyclooctadiene)rhodium (I) tetrafluoroborate and [(R)-(+)-2,2'-bis(diphenylphosphino)1,1'-binaphthyl]dichlororuthenium (II), and wherein the ligand is selected from (S)-(+)-1-((R)-2-diphenylphosphino)ferrocenyl)ethyldi-tert-butylphosphine, (S)-(-)-1-((R)-2-diphenylphosphino)ferrocenyl)ethyldi-tert-butylphosphine, (S)-NMDPP, (R)-(+)-BINAP, (R,R)-(2-(4'-isopropyloxazolin-2'-yl)ferrocenyl)diphenylphosphine, (R)-(S)-JOSIPHOS, (R)-(S)-Cy2PF-P(tBu)2, (R)-(S)-PPF-P(tBu)2, (S,S)-Ph-BPE, (S, S)-Me-BPE, (S,S)-DIOP, (S)-MONOPHOS, cane ligand (S,S)-DACH-phenyl, (R)-SEGPHOS, (S)-MeO-BIPHEP, (S,S)-TsDPEN, (R,R)-f-SpiroPhos and (R,R)-Et-Duphos. 2. The method according to claim 1, wherein the coenzyme comprises a NADP salt and/or a NAD salt, preferably a NADP salt; preferably the glucose dehydrogenase comprises the amino acid sequence presented under SEQ ID No: 1. 3. The method according to claim 1, wherein the imine reductase comprises the amino acid sequence presented under SEQ ID No: 2-4, SEQ ID No: 12. 4. The method according to claim 1, wherein the catalytic reduction reaction using a biocatalytic method is carried out at a temperature in the range from 15 to 45°C; preferably, the catalytic reduction reaction is carried out in a buffer solution; and the buffer solution comprises a phosphate buffer, a Tris-HCl based buffer or a TEA-HCl based buffer; Preferably, the catalytic reduction reaction is carried out at a pH in the range of 6.0 to 8.0. 5. The method according to claim 1, wherein the catalytic reduction reaction using a chiral metal catalyst is carried out at a temperature in the range from 50 to 100°C; preferably, during the catalytic reduction reaction, the pressure of hydrogen gas is maintained in the range of 1.0 to 6.0 MPa; Preferably, the catalytic reduction reaction is carried out at a pH in the range of 4.0 to 13.0. 6. The method according to any one of claims 1 to 5, wherein the alkene compound represented by formula I or the iminium cationic compound represented by formula II is obtained by desalting and/or cyclizing a compound represented by formula III or a salt thereof, Formula III; preferably the salt comprises hydrochloride, dihydrochloride, hydrobromide, dihydrobromide, sulfate or bisulfate. 7. The method according to claim 6, wherein the method for synthesizing a salt of a compound represented by formula III comprises: mixing compound A with N-methylpyrrolidone and an organic base to obtain compound D and mixing compound D with an acid to obtain a salt of the compound of formula III; and the reaction equations are as follows: 8. The method according to claim 6, wherein the method for synthesizing a salt of a compound represented by formula III comprises: mixing compound A with N-methylpyrrolidone and an organic base and neutralizing with an acid to achieve a pH of 7-8, thereby obtaining compound B, and mixing compound B with an acid to obtain a salt of a compound represented by formula III; and the reaction equations are as follows: 9. The method according to claim 7 or 8, wherein the method for synthesizing compound A comprises mixing nicotinic acid with methanol and esterifying in a highly acidic medium to thereby obtain compound A. 10. The method according to any one of claims 1-9, wherein the method for synthesizing (S)-nicotine comprises: mixing nicotinic acid with methanol and esterifying in a highly acidic medium to thereby obtain compound A; mixing compound A with N-methylpyrrolidone and an organic base, optionally adding a first acid for neutralization to thereby obtain compound B or compound D; mixing compound B or compound D with an acid to thereby obtain a salt of the compound represented by formula III; desalting and cyclizing the salt of the compound represented by formula III, and reducing the resulting product using a biocatalytic method or a chiral metal catalyst to obtain (S)-nicotine; and the reaction equations are as follows:

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