CN104830924B - Carbonyl reduction enzymes biocatalysis produces thienyl chirality alcohol compound - Google Patents
Carbonyl reduction enzymes biocatalysis produces thienyl chirality alcohol compound Download PDFInfo
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Abstract
The invention discloses the method that using carbonyl reductase as biocatalyst, corresponding (S) configuration alcohol is synthesized through 5 kinds of thienyl ketone substrates of asymmetric reduction.The catalyst of this method is easily prepared, and aqueous catalysis, reaction condition is gentle, and reaction system is simple, no side reaction, and ee values are most to be more than 99%, provides environmental-friendly Biocatalysis method for the production of thienyl chirality alcohols, and have commercial application potentiality.
Description
Technical field
The invention belongs to enzyme and biocatalysis field, and in particular to produce thienyl hand using carbonyl reduction enzymes biocatalysis
The method of property alcohol compound.
Background technology
Duloxetine (Duloxetine) is third generation antidepressants, is one of most important antidepressants of market sale,
Can effectively inhibit 5- hydroxyl color ammonia and norepinephrine reuptake (Wong, D.T., et al.Life Sci, 1988,43：
2049-2057), and to diseases such as overall pain, gastrointestinal disturbances also there is good drug effect.Research shows (S)-Duloxetine
(chemical name, (S)-(+)-N- methyl -3- (1- naphthoxys) -3- (2- thiophene)-propylamine) just has pharmaceutical activity, therefore (S) structure
The acquisition of type intermediate becomes the committed step in Duloxetine synthesis.
At present also using chiral resolution is carried out after chemical technology reduction, it is mainly deposited for the production of Duloxetine chiral intermediate
The problem of have：Substantial amounts of resolving agent is needed, reactions steps are grown, and energy consumption is big；Waste discharge amount is big, and environmental pollution is serious；Yield
Low, optical purity of products is not high, heavy-metal residual problem.Based on living things catalysis stereoselectivity is high, accessory substance is few, reaction condition
Gentle and environmental-friendly etc. unique advantage, is also sent out rapidly using the research of living things catalysis production duloxetine intermediate
Exhibition.
The structure of (S)-Duloxetine is analyzed, the principle such as is easily catalyzed, stablizes according to inverse composition strategy and substrate, can be with
Using 5 kinds of thienyl ketone substrate S1-S5 listed below Duloxetine chiral intermediate can be obtained through asymmetric reduction
(Tang, C.G., et al.Biotechnol Lett, 2011,33 (7)：1435-1440).
The living things catalysis research of these thienones is concentrated mainly on substrate S1, S2 and S5, and there is presently no life by S3 and S4
The report of thing catalysis.The main level of the document report of thienone S1, S2 and S5 living things catalysis is as follows：
(1) substrate S1：Concentration of substrate 10g/l, 6h reaction terminates, product ee values>98% (Wada, M., et al.Biosci
Biotech Bioch, 2004,68 (7)：1481-1488).
(2) substrate S2：Ni6sh is answered in bottom with the anti-ti of dense do the degree r1or0ugl/aL, glu of thing Rho, and complete thin production born of the same parents' thing is urged for ee>
Change 98 doses of %, concentration of substrate 30g/l, react 48h, conversion ratio>95%, ee value>99% (Tang, C.G., et
Al.Biotechnol Lett, 2011,33 (7)：1435-1440)；Using ChKRED15 crude enzyme liquids as biocatalyst, in 2h
The substrate of 20g/l can be converted, conversion ratio is more than 99.9% (CN103740738A) for more than 99%, ee values.
(3) substrate S5：Using Candida tropicalis as biocatalyst, concentration of substrate 1g/l, reacts 60h, product
Ee values>99% (Soni, P., et al.App Microbiol Biotechnol, 2005,67 (6)：771-777)；With alcohol dehydrogenase
Enzyme ADH-LK is biocatalyst, and concentration of substrate 100g/l, reacts 24h, product ee values>99% (Codexis,
INC.WO2010025238A2)。
Although researchers have screened multiple-microorganism bacterial strain (or carbonyl reductase) to realize above-mentioned S1, S2 and S5 bottom
The living things catalysis of thing, but all things considered, enzyme source is still less, and most catalytic process are using original bacteria as biocatalyst；Meanwhile
Substrate feed concentrations are also universal relatively low (only S5 have patent report feed intake achievable 100g/l), also have relatively large distance from application.
The more new excellent enzyme sources of active development, will establish solid base to realize that the living things catalysis of thienyl chirality alcohol compound synthesizes
Plinth.
The content of the invention
The object of the present invention is to provide one kind using carbonyl reductase as biological catalyst preparation thienyl chirality alcohols chemical combination
The method of thing.Specifically, the substrate used in the present invention is following general structure (I), utilizes different efficient high selectivity carbonyls
Reductase is biocatalyst, and corresponding (S) configuration chiral alcohol is obtained through asymmetric reduction.
Living things catalysis process can represent as follows：
Wherein, R substituent can be ester-containing structure, halogen-substituted alkyl or containing amino structure etc.；
Further, R substituent COOCH3, COOCH2CH3, CONHCH3, CH2Cl, CN or CH2N(CH3)2, these substrates
S configuration alcohol is obtained through carbonyl reduction enzymes biocatalysis, the chiral intermediate in being synthesized as Duloxetine.
Carbonyl reductase tool box of the biocatalyst of the present invention from this experimental construction.According to the public affairs of this area
Knowledge, those skilled in the art can obtain biocatalyst in the following manner altogether.First, the information (gene of carbonyl reductase
Sequence and amino acid sequence) (http can be inquired about by the NCBI accession number of the enzyme://www.ncbi.nlm.nih.gov/) or
From sequence (the SEQ ID in specification sequence table：No.1-4 know in), then synthesize and obtain through gene chemical synthesis company, and with normal
Technological means structure coli expression system is advised, following enzymes are obtained as biocatalyst through overexpression.
Most suitable substrate of the invention and corresponding carbonyl reductase are as follows：
(1)
The carbonyl reductase of catalysis substrate S1 has：(gene order is SEQ ID to KRED2157：No.1, amino acid sequence are
SEQ ID：No.2), ChKRED07 (NCBI accession number：KC342007), ChKRED10 (NCBI accession number：KC342010),
ChKRED11 (NCBI accession number：KC342011), ChKRED12 (NCBI accession number：) or ChKRED14 (NCBI KC342012
Accession number：KC342014).
The homologue S1-1 of substrate S1, its structural formula areThe above-mentioned of S1 can be catalyzed in the present invention
Carbonyl reductase can equally be catalyzed the compound, obtain corresponding S configurations alcohol.
(2)
The carbonyl reductase of catalysis substrate S2 has：ChKRED07 (NCBI accession number：KC342007), KRED2157 (genes
Sequence is SEQ ID：No.1, amino acid sequence are SEQ ID：No.2), ChKRED03 (NCBI accession number：KC342003),
ChKRED10 (NCBI accession number：KC342010), ChKRED23 (NCBI accession number：KC342023), (NCBI is logged in ChKRED24
Number：KC342024), ChKRED26 (NCBI accession number：) or ChKRED14 (NCBI accession number KC342026：KC342014).
(3)
The carbonyl reductase of catalysis substrate S3 has：ChKRED07 (NCBI accession number：) or KRED2935 (bases KC342007
Because sequence is SEQ ID：No.3, amino acid sequence are SEQ ID：No.4).
(4)
The carbonyl reductase of catalysis substrate S4 has：ChKRED07 (NCBI accession number：KC342007), ChKRED10 (NCBI
Accession number：KC342010), ChKRED11 (NCBI accession number：KC342011), ChKRED12 (NCBI accession number：KC342012),
ChKRED14 (NCBI accession number：KC342014), (gene order is SEQ ID to KRED2157：No.1, amino acid sequence SEQ
ID：No.2), ChKRED03 (NCBI accession number：KC342003) or KRED2935 (gene order is shown in SEQ ID：No.3, amino
Acid sequence is shown in SEQ ID：No.4).
(5)
The carbonyl reductase of catalysis substrate S5 has：ChKRED10 (NCBI accession number：KC342010), ChKRED03 (NCBI
Accession number：) or ChKRED05 (NCBI accession number KC342003：KC342005).
The living things catalysis system and reaction condition of the present invention：
(1) when biocatalyst is resting cell, living things catalysis system is：Phosphate buffer (0.1M, pH6~8), carbonyl
Base reductase recombinant bacterium resting cell, general structure (I) substrate and glucose.Resting cell concentration is 50~300g/l (with wet
Thalline meter)；Final concentration of 1~the 100g/l of general structure (I) substrate；The concentration of glucose is 1%~5% (w/v).
Substrate, which is first dissolved in DMSO, is configured to 30~50% (w/v) storage liquid.
(2) when biocatalyst is the pure enzyme of carbonyl reductase or thick enzyme, living things catalysis system is：
Phosphate buffer (0.1M, pH6~8), carbonyl reductase, general structure (I) substrate and reduced coenzyme NAD
(P)H；
Alternatively, living things catalysis system is：
Phosphate-buffered (0.1M, pH6~8), carbonyl reductase, general structure (I) substrate, oxidized coenzyme NAD (P)+,
Glucose dehydrogenase and glucose.
The enzyme amount of carbonyl reductase can be adjusted according to concentration of substrate, and preferably concentration is pure 0.1~10g/l of enzyme, and thick enzyme 1~
20g/l (with total protein gauge)；Final substrate concentrations are 1~150g/l；NAD(P)+0.1~1g/l of concentration；Glucose dehydrogenase is dense
Spend 2U/ml~50U/ml；The concentration of glucose is 5%~15% (w/v).
Living things catalysis condition：20~35 DEG C, 50~220rpm of rotating speed of temperature, transformation time are 1~24h.
Beneficial effects of the present invention：
The invention discloses with carbonyl reductase Biocatalysis method obtain thienyl chirality alcohol compound, this method
Catalyst is easily prepared, and aqueous catalysis, reaction condition is gentle, and reaction system is simple, no side reaction, and the ee value overwhelming majority is more than
99%, provide selectable environmental-friendly Biocatalysis method for the production of thienyl chirality alcohols.Carbonyl of the present invention
Base reductase is more, and abundant enzyme source is provided for this kind of compound living things catalysis.
The chiral intermediate P1-P5 that the present invention is obtained can be as the intermediate in Duloxetine synthesis.To produce P1
Exemplified by product, when ChKRED12 is catalyzed substrate S1, when concentration of substrate is 100g/l, conversion ratio>99%, ee value>99%, have very
Strong commercial application potentiality.
It should be noted that according to this area common knowledge, due to the degeneracy of the codon of nucleotide, every above-mentioned carbonyl
Base reductase is through allelic mutation or with one or more amino acid addition, insertion, missing or/and substitutions and with urging
The enzyme for changing corresponding (S) the configuration alcohol of structure formula (I) substrate generation belongs to protection scope of the present invention.
Embodiment
The present invention is explained with reference to embodiments.Embodiment is for ease of being better understood from the present invention, but not
Limitation of the present invention.
Embodiment 1：The screening of carbonyl reductase
In method well known in the art, carbonyl reduction enzyme gene is connected into pET28a (+) carrier, is transferred to Escherichia coli
Heterogenous expression in BL21-DE3, obtains resting cell as biocatalyst.During screening, living things catalysis and system are：Phosphate
Buffer solution (0.1M, pH7.0), carbonyl reductase recombinant bacterium resting cell 150g/l (in terms of wet thallus), substrate 1g/l and grape
2% (w/v) of sugar.Reaction time is 24h, 30 DEG C of reaction temperature, rotating speed 200rpm.
Meanwhile pET28 (+) empty carrier for not being connected into carbonyl reduction enzyme gene is equally transferred to e. coli bl21-DE3
Middle heterogenous expression, all kinds of substrates are converted by blank control of this resting cell.The selection result presented below, blank control is not
Can conversion of substrate.
After reaction, extracted 3 times with isometric ethyl acetate, merge organic phase, add anhydrous sodium sulfate drying, then
Vacuum rotary steam removes solvent, adds isopropanol (HPLC grades) sample dissolution, after high speed centrifugation the conversion ratio of HPLC determination samples and
Product ee values.
(1) carbonyl reduction enzymatic substrate S1：
Conversion ratio detects：SIL-100A(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：hexane:
Isopropanol=70:30；Flow velocity：0.4ml/min；Appearance time：Substrate 9.3min, product 10.1min.
Product ee values detect：Daicel CHIRALCEL AD-H (4.6 × 250mm) column temperature：35℃；Mobile phase (v/v)：
hexane:Isopropanol=98:2；Flow velocity：0.8ml/min；Appearance time：S types 39.0min, R type 40.5min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S1 see the table below 1：
Table 1
(2) carbonyl reduction enzymatic substrate S1-1：
Conversion ratio detects：SIL-100A(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：hexane:
Isopropanol=70:30；Flow velocity：0.4ml/min；Appearance time：Substrate 9.9min, product 11.2min.
Product ee values detect：Daicel CHIRALCEL AS-H (4.6 × 250mm) column temperature：35℃；Mobile phase (v/v)：
hexane:Isopropanol=95:5 flow velocitys：0.8ml/min；Appearance time：S types 42.2min, R type 43.3min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S1 see the table below 2：
Table 2
Since substrate S1 and S1-1 structure is much like, the carbonyl reductase that can convert both is identical, simply conversion ratio
It is variant.
(3) carbonyl reduction enzymatic substrate S2：
Conversion ratio measures：SIL-100A(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：hexane:
Isopropanol=70:30；Flow velocity：0.8ml/min；Appearance time：Substrate 12.0min, product 8.9min.
Product ee values detect：Daicel CHIRALCEL OJ-H(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：
hexane:Isopropanol=90:10；Flow velocity：0.5ml/min；Appearance time：S types 22.8min, R type 24.2min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S2 see the table below 3：
Table 3
(4) carbonyl reduction enzymatic substrate S3：
Conversion ratio detects：SIL-100A(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：hexane:
Isopropanol=70:30；Flow velocity：0.4ml/min；Appearance time：Substrate 8.9min, product 9.8min.
When product ee values detect：Daicel CHIRALCEL AS-H(4.6×250mm)；Column temperature：35℃；Mobile phase (v/
v)：hexane:Isopropanol=97:3；Flow velocity：0.8ml/min；Appearance time：S types 21.6min, R type 23.7min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S3 see the table below 4：
Table 4
(4) carbonyl reduction enzymatic substrate S4：
Conversion ratio detects：SIL-100A(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：hexane:
Isopropanol=70:30；Flow velocity：0.8ml/min；Appearance time：Substrate 7.8min, product 5.3min.
Product ee values measure：Daicel CHIRALCEL OJ-H(4.6×250mm)；Column temperature：35℃；Mobile phase (v/v)：
hexane:Isopropanol=80:20；Flow velocity：0.8ml/min；Appearance time：S types 21.0min, R type 23.7min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S4 see the table below 5：
Table 5
(5) carbonyl reduction enzymatic substrate S5：
Conversion ratio detects and product ee value testing conditions：Daicel CHIRALCEL OJ-H(4.6×250mm)；Column temperature：
35℃；Mobile phase (v/v), hexane:isopropanol:Triethylamine=95:5:1.5；Flow velocity：0.8ml/min；Go out
Peak time：Substrate 11.2min, S type product 13.5min, R type 12.2min.
The carbonyl reductase and its conversion ratio and ee values for being capable of conversion of substrate S5 see the table below 6：
Table 6
Embodiment 2：The structure of efficient coenzyme circulating system
When making catalyst with pure enzyme or thick enzyme, glucose dehydrogenase and glucose structure coenzyme circulating system can be added.
Coenzyme circulating system can be expressed as (substrate is by taking S2 as an example)：
Glucose dehydrogenase is bought in Sigma (CAS using the efficient glucose dehydrogenase of commercialization：9028-53-
9).To ensure that the efficient of reaction carries out, the supply of coenzyme must be sufficient.Concentration of glucose is 10% (w/v) in reaction system, Portugal
Grape glucocorticoid dehydrogenase is 20U/ml.With the progress of reaction, the amount of gluconic acid is progressively accumulated, and reaction system pH declines therewith.For
Reaction is set efficiently persistently to carry out, the pH of reaction system should be controlled between 6.5-8.0,10%NaOH is used in practical application
Aqueous solution takes the mode of interval to adjust pH.
Embodiment 3：Carbonyl reductase ChKRED12 crude enzyme liquids catalysis 10g/l substrates S1
Take fresh cultured carbonyl reductase ChKRED12 recombinant bacterium wet thallus be resuspended in phosphate buffer (0.1M,
PH8.0), with homogenizer smudge cells, 13,000rpm, 4 DEG C of centrifugation 20min, gained supernatant is crude enzyme liquid.
Kaliumphosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED12 concentration 10mg/ml (crude enzyme liquid), substrate S1
Concentration 10g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP+Concentration 0.2mM, 30 DEG C, transformation time 2h, reacted
A pH to 8.0 is adjusted in journey per 30min.Isometric ethyl acetate extraction terminates reaction, sample treatment and detection method
It is identical with embodiment 1 (1).The result shows that 10g/l substrates can just convert completely in 2h, and the ee values of product are more than 99.9%.
Embodiment 4：Carbonyl reductase ChKRED12 crude enzyme liquids catalysis 20g/l substrates S1
In addition to S1 concentration of substrate increases to 20g/l, remaining reaction system is same as Example 3, transformation time 5h, reaction
During per 30min adjust a pH to 8.0.Isometric ethyl acetate extraction terminates reaction, sample treatment and detection side
Method is identical with embodiment 1 (1).The result shows that 10g/l substrates can just convert completely in 5h, and the ee values of product are more than 99.9%.
Embodiment 5：Carbonyl reductase ChKRED12 crude enzyme liquids catalysis 50g/l substrates S1
In addition to S1 concentration of substrate increases to 50g/l, remaining reaction system is same as Example 3, transformation time 12h, instead
A pH to 8.0 is adjusted during answering per 30min.Isometric ethyl acetate extraction terminates reaction, sample treatment and detection
Method is identical with embodiment 1 (1).The result shows that 50g/L substrates can realize complete conversion in 12h, and the ee values of product are more than
99.9%.
Embodiment 6：Carbonyl reductase ChKRED12 crude enzyme liquids catalysis 100g/l substrates S1
In addition to S1 concentration of substrate increases to 100g/l, remaining reaction system is same as Example 3, transformation time 24h, instead
A pH to 8.0 is adjusted during answering per 30min.Isometric ethyl acetate extraction terminates reaction, sample treatment and detection
Method is identical with embodiment 1 (1).The result shows that 100g/l substrates S1 can realize complete conversion in 24h, and the ee values of product are more than
99.9%.
Embodiment 7：Carbonyl reductase KRED2157 crude enzyme liquids catalysis 10g/l substrates S2
Take fresh cultured carbonyl reductase KRED2157 recombinant bacterium wet thallus be resuspended in phosphate buffer (0.1M,
PH8.0), with homogenizer smudge cells, 13,000rpm, 4 DEG C of centrifugation 20min, gained supernatant is crude enzyme liquid.
Kaliumphosphate buffer (0.1M, pH8.0), carbonyl reductase KRED2157 concentration 10mg/ml (crude enzyme liquid), substrate S2
Concentration 10g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP+Concentration 0.2mM, 30 DEG C, transformation time 2h, reacted
A pH to 8.0 is adjusted in journey per 2h.Isometric ethyl acetate extraction terminates reaction, sample treatment and detection method and reality
It is identical to apply example 1 (3).The result shows that 10g/l substrates S2 can just convert conversion completely in 24h, and the ee values of product are more than 99.9%.
Embodiment 8：Carbonyl reductase ChKRED07 crude enzyme liquids catalysis 20g/l substrates S3
The carbonyl reductase ChKRED07 recombinant bacterium wet thallus of fresh cultured is taken to be resuspended in phosphate buffer (0.1M, pH
8.0), with homogenizer smudge cells, 13,000rpm, 4 DEG C of centrifugation 20min, gained supernatant is crude enzyme liquid.
Kaliumphosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED07 concentration 10mg/ml (crude enzyme liquid), substrate S3
Concentration 20g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP+Concentration 0.2mM, reacts by 30 DEG C, transformation time 24h
During per 2h adjust a pH to 8.0.Isometric ethyl acetate extraction terminate reaction, sample treatment and detection method with
Embodiment 1 (4) is identical.The result shows that conversion ratio is more than 99%, and the ee values of product are more than 99.9%.
Embodiment 9：Carbonyl reductase ChKRED07 crude enzyme liquids catalysis 20g/l substrates S4
The carbonyl reductase ChKRED07 recombinant bacterium wet thallus of fresh cultured is taken to be resuspended in phosphate buffer (0.1M, pH
8.0), with homogenizer smudge cells, 13,000rpm, 4 DEG C of centrifugation 20min, gained supernatant is crude enzyme liquid.
Kaliumphosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED07 concentration 10mg/ml (crude enzyme liquid), substrate S4
Concentration 20g/l, GDH concentration 20U/ml, glucose 10% (w/v), NADP+Concentration 0.2mM, reacts by 30 DEG C, transformation time 24h
During per 2h adjust a pH to 8.0.Isometric ethyl acetate extraction terminate reaction, sample treatment and detection method with
Embodiment 1 (5) is identical.The result shows that conversion ratio is more than 99%, and the ee values of product are more than 99.9%.
Claims (1)
1. a kind of preparation method of S configurations alcohol, it is characterised in that asymmetric through carbonyl reductase with general structure (I) for substrate
Living things catalysis obtains S configuration alcohol,
Wherein R substituent is COOCH3, carbonyl reductase used is ChKRED12, its amino acid sequence NCBI accession number is：
KC342012；R substituent is CH2Cl or CN, carbonyl reductase used are ChKRED07, its amino acid sequence NCBI accession number
For：KC342007.
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| WO2010025238A2 (en) * | 2008-08-27 | 2010-03-04 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
| CN103740738A (en) * | 2014-01-12 | 2014-04-23 | 中国科学院成都生物研究所 | Carbonyl reductase, gene thereof, and application thereof in preparing Duloxetine chiral intermediates |
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| WO2010025238A2 (en) * | 2008-08-27 | 2010-03-04 | Codexis, Inc. | Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
| CN103740738A (en) * | 2014-01-12 | 2014-04-23 | 中国科学院成都生物研究所 | Carbonyl reductase, gene thereof, and application thereof in preparing Duloxetine chiral intermediates |
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