CN103740738A - Carbonyl reductase, gene thereof, and application thereof in preparing Duloxetine chiral intermediates - Google Patents
Carbonyl reductase, gene thereof, and application thereof in preparing Duloxetine chiral intermediates Download PDFInfo
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Abstract
The invention discloses a carbonyl reductase (i)Ch (/i) KRED15 derived from Chryseobacterium ((i) Chryseobacterium (/i) (i) (/i) sp. CA49), and a coding gene thereof; the invention also discloses application of the carbonyl reductase as a biocatalyst in preparing a Duloxetine chiral intermediate ((i) S (/i))-N-methyl-3-hydroxy-3-(2-thienyl) propanamide. The enantiomer excess of products is greater than 99.9%. 20 g/L of substrate can be catalyzed by Ch(/i) KRED15 crude enzyme (2U/mL), and the conversion rate of over 99% can be obtained in 2 hours; a coenzyme circulation system is simple, and has large industrial application prospect.
Description
Technical field
The present invention relates to a kind of new gene and protein thereof, be specifically related to one and derive from novel carbonyl reductase ChKRED15 and the gene thereof of Chryseobacterium sp (Chryseobacterium sp.CA49), and utilize this carbonyl reductase to prepare duloxetine chiral intermediate as biological catalyst, belong to using microbe and enzyme engineering field.
Background technology
Duloxetine is third generation thymoleptic, the topmost thymoleptic of market sale, can effectively suppress re-uptake (Wong DT, et al.Life Sci, 1988 of 5-hydroxyl look ammonia and norepinephrine, 43:2049-2057), efficiently, safety, side effect is little, to other symptoms as whole body pain and also effective in cure (Bymaster FP, et al.Neuropsychopharmacology of gastrointestinal disturbance, 2001,25:871-880).This medicine is also approved for neuralgia that diabetes cause and the treatment (Norton PA, et al.Am J Obstet Gynecol, 2002,187:40-48) of female incontinence.
Duloxetine contains a chiral centre, and research only shows that the enantiomorph of (S) configuration has pharmaceutical activity.(S) the synthetic committed step of-duloxetine is the acquisition of (S) configuration chiral alcohol intermediate.At present to the acquisition of (S) configuration chiral alcohol intermediate mainly by chemistry route, but there are problems, as chemistry splits, need to use a large amount of resolving agents, reactions steps is long, the high and waste discharge amount of energy consumption is large etc.; Based on transition metal chiral ligand catalytic hydrogenation system, because optical purity of products is not high and the problem such as heavy-metal residual, cause it to be difficult to be applied to the production of medicine intermediate.
Compared with chemical process, biocatalysis process has chemistry, the regio-and stereo-selectivity of height, reaction conditions gentleness, and aftertreatment is easy, and energy consumption is lower, is a kind of eco-friendly synthetic method, is also that current Chemical Manufacture and association area develop a megatrend.Wherein biocatalysis asymmetric reduction has developed into the important component part of research and development department of global Ge great pharmaceuticals.But; overwhelming majority research up to now concentrates on the substrate that phenyl ketone is skeleton; and the research of substrate take hetero-aromatic ring ketone as skeleton is more rare; wherein especially very few (the Wada M synthetic relevant to duloxetine chiral intermediate; et al.Biosci Biotechnol Biochem; 2004,68:1481-1488; St ü rmer R, et al., 2008, US0318288A1; Tang CG, et al.Biotechnol Lett, 2011,33:1435-1440).Wherein, (the Tang CG such as Tang Chuangen, et al. Biotechnol Lett, 2011,33:1435-1440) within 2011, reported that substrate N-methyl-3-carbonyl-3-(2-thienyl) propionic acid amide that using sticky red rhodotorula (Rhodotorula sp.CY12) catalysis hetero-aromatic ring ketone is skeleton generates (S)-dulouxetine key alcohol intermediate synthetic route, this technique is take original bacterium resting cell as biological catalyst, in 48h, can conversion of substrate concentration be 30g/l, for (S)-dulouxetine key alcohol intermediate provides one can system of selection.For the how good catalyzer of this substrate screening can further enrich biocatalysis work box, excavate how potential alternation enzyme source.
Summary of the invention
The invention discloses carbonyl reductase ChKRED15 and gene thereof that one derives from Chryseobacterium sp (Chryseobacterium sp.CA49), and provide the construction process of this enzyme heterogenous expression system and this enzyme as biological catalyst the method for the preparation of duloxetine chiral intermediate.
The nucleotide sequence (738bp) of the gene of carbonyl reductase ChKRED15 is shown in SEQ ID No.1.
The aminoacid sequence of the protein (245aa) of carbonyl reductase ChKRED15 genes encoding is shown in SEQ ID No.2.
Carbonyl reductase ChKRED15 clones acquisition from Chryseobacterium sp (Chryseobacterium sp.CA49) genome, the deposit number of this bacterium is CCTCC M2012484(Wuhan, China, Chinese Typical Representative culture collection center, preservation date is on November 27th, 2012).
By designing following primer: forward: 5 '-GCG
gAATTCaTG AAA ACA GTA TTA ATT ACA GGC GCC-3 ' (restriction enzyme site: EcoR I); Reverse: 5 '-GCG
aAGCTTcTA CCA CGG ACT GAT TCC GG-3 ' (restriction enzyme site: Hind III), take Chryseobacterium sp (Chryseobacterium sp.CA49) genomic dna as template, through pcr amplification, obtain target gene, the protein called after carbonyl reductase ChKRED15 of this genes encoding.Target gene is connected into pET28a (+) carrier, and proceeds to E.coli BL21(DE3), build pET28a (+)-BL21 heterogenous expression system.After heterogenous expression product is purified, obtain pure enzyme.
According to existing common knowledge, any gene is connected into all kinds of expression vectors after operation or transformation, is converted into suitable host cell, all can overexpression target protein through felicity condition induction.Therefore, the carrier that carbonyl reductase ChKRED15 expresses can be pET or pCW or pUC or pPIC9k etc., and expressive host can be intestinal bacteria, pichia spp, streptomycete etc.
The present invention also provides carbonyl reductase ChKRED15 at conversion of substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide, to generate the application in (S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide as biological catalyst.In above-mentioned application, described catalytic condition can be selected by the normal condition of this type of reaction of this area.When carbonyl reductase ChKRED15 makes catalyzer, reaction system is: carbonyl reductase ChKRED15, potassium phosphate buffer or Tris-HCl damping fluid, NADH or NADPH, substrate N-methyl-3-carbonyl-3-(2-thienyl) propionic acid amide is (because the solubleness of substrate in water is lower, therefore be first dissolved in the substrate stock solution that is made into 40% in DMSO, then join in reaction system w/v).Enzyme concn, NADH or NADPH amount, the concentration of substrate etc. of reaction system can be adjusted.After reaction finishes, ethyl acetate extraction termination reaction, obtains product.This endonuclease capable highly selective of empirical tests is the carbonyl asymmetric reduction of catalyzing N-methyl-3-carbonyl-3-(2-thienyl) propionic acid amide (ee>99%).
Can carry out comprising the pure enzyme of carbonyl reductase ChKRED15, corresponding recombinant bacterium resting cell, crude enzyme liquid or thick enzyme powder etc. other existing form of above-mentioned biocatalytic reaction.While adopting carbonyl reductase ChKRED15 recombinant bacterium resting cell to make catalyzer, conventionally with coenzyme circulation substrate, replace coenzyme to react, conventional coenzyme circulation substrate is: glucose and/or Virahol.
According to existing common knowledge, any gene can change its sequence by DNA mutation technology, thereby produces various mutant, and the expressed protein of these mutant often has identical function.The present invention relates to gene and product thereof and also there is identical feature.Through allelic mutation or through adding, insert, disappearance is or/and replace aminoacid sequence that one or more amino acid and coding have equal activated protein and can serve as equally biological catalyst conversion of substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide and generate (S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide.
The carbonyl reductase ChKRED15 the present invention relates to can generate S-alcohol by Efficient Conversion N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide, and alternative novel enzyme source is provided for (S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide biocatalysis is synthetic.
The potentiality of carbonyl reductase ChKRED15: this albumen is made crude enzyme liquid in intestinal bacteria after overexpression, 2U/mL enzyme amount just can transform the substrate of 20g/L in 2h, and transformation efficiency is more than 99%, and ee value is greater than 99.9%.Crude enzyme liquid conversion process is simple, and coenzyme circulating system is stable, has larger prospects for commercial application.
Accompanying drawing explanation
SDS-PAGE figure after Fig. 1 carbonyl reductase ChKRED15 albumen overexpression, M is marker, 1:E.coli(pET28a) empty carrier, 2:E.coli(pET28a-ChKRED15) full cell, 3:E.coli(pET28a-ChKRED15) supernatant after cytoclasis, after 4:ChKRED15 purifying.
embodiment
Below in conjunction with embodiment, explain the present invention.Embodiment is for ease of better understanding the present invention, but limitation of the present invention not.
The amplification of embodiment 1 carbonyl reductase (ChKRED15) gene
According to Chryseobacterium sp (Chryseobacterium sp.CA49) genome sequencing information, excavate wherein a large amount of carbonyl reductases, one of them enzyme with catalyzing N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide generation (S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide function is the carbonyl reductase ChKRED15 the present invention relates to, and this gene acquisition process is conventional working method.Design following primer: forward: 5 '-GCG
gAATTCaTG AAA ACA GTA TTA ATT ACA GGC GCC-3 ' (restriction enzyme site: EcoR I); Reverse: 5 '-GCG
aAGCTTcTA CCA CGG ACT GAT TCC GG-3 ' (restriction enzyme site: Hind III), take Chryseobacterium sp CCTCC M2012484 genomic dna as template, obtains carbonyl reductase ChKRED15 gene through pcr amplification.
PCR program is: 95 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, last 72 ℃ are extended 10min.
PCR product is reclaimed to test kit through sepharose after with 0.8% agarose electrophoresis and reclaim object fragment (day root biochemical technology Beijing company limited), then to be connected to pET28a (+) carrier with identical restriction enzyme site after EcoR I/Hind III double digestion, obtain recombinant plasmid pET28a-ChKRED15, and deliver to the order-checking of Shanghai Sheng Gong bio-engineering corporation.Through order-checking, target gene total length 738bp, its base sequence is as shown in sequence table SEQ ID No.1.
By the correct plasmid chemical conversion of order-checking to E.coli BL21(DE3) competent cell, obtain recon E.coli(pET28a-ChKRED15).
The abduction delivering of embodiment 2 carbonyl reductases (ChKRED15)
The recon E.coli(pET28a-ChKRED15 that picking embodiment 1 is constructed) mono-clonal, be inoculated in containing in the LB substratum of 50 μ g/mL kantlex, in 37 ℃, 180rpm cultivates 16h as seed liquor, with 1% rate of vaccination, be forwarded to (500mL capacity shaking flask dress liquid 200mL substratum) in fresh LB or TB substratum, in 37 ℃ of shaking culture 4h, adding subsequently final concentration is the expression of the IPTG induction carbonyl reductase ChKRED15 gene of 0.5mM, after 30 ℃ of induction 36h, 4 ℃, 8000rpm frozen centrifugation 10min gathers in the crops thalline.
Thalline can be used as biological catalyst or for protein purification.
The separation and purification of embodiment 3 carbonyl reductases (ChKRED15)
By in embodiment 2 obtain thalline with binding buffer liquid (100mM, pH8.0 sodium phosphate buffer, containing 300mM NaCl, 5mM imidazoles) resuspended after, through high-pressure homogeneous crusher machine, the centrifugal 15min of 12000rpm, after supernatant is hatched with the Ni affinity chromatography resin of crossing through above-mentioned combination liquid balance, use rinsing damping fluid (100mM, pH8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazoles) rinsing is to substantially without foreign protein, subsequently with elution buffer (100mM, pH8.0 sodium phosphate buffer, containing 300mM NaCl, 250mM imidazoles) wash-out collect target protein, electrophoresis is identified and is merged target protein after purity and with dialysis buffer liquid (100mM, pH8.0 potassium phosphate buffer) dialysis 48h, after ultrafiltration and concentration, utilizing BCA Protein Assay kit to measure protein concentration is 8mg/mL, it is 5mg/mL packing that enzyme liquid is diluted to final concentration, frozen in-80 ℃ (carbonyl reductase ChKRED15 protein electrophoresis figure is shown in accompanying drawing 1).
The determination of activity of embodiment 4 carbonyl reductases (ChKRED15)
The pure enzyme of carbonyl reductase ChKRED15 that separation and purification in embodiment 3 is obtained is for catalytic substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide.
Reaction system 10mL, comprises potassium phosphate buffer (0.1M, pH8.0), pure enzyme concn 0.1g/L, substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 1g/L, NADPH10mM.30 ℃, rotating speed 50rpm, transforms 30min.Reaction finishes rear by the long-pending ethyl acetate extracting twice of monoploid, merge organic layer and use anhydrous sodium sulfate drying, after blowing, Nitrogen evaporator nitrogen removes ethyl acetate, gained resistates fully dissolves with 0.1mL Virahol (HPLC level), get 2 μ l samples, HPLC detects transformation efficiency and ee value (detection method is shown in embodiment 5).Calculating transformation efficiency is 66%, ee value >99.9%(S type).In following examples, product is S configuration.
Substrate and product data are as follows:
N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide data are as follows:
White powder
1H?NMR[(600MHz,CDCl
3)δppm2.86(d,3H,-NMe),3.8952(s,2H,-CH
2),7.12(m,2H,aromatic?and?NH),7.74(d,1H,aromatic),7.836(d,1H,aromatic)]。
(S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide data are as follows:
White powder, [α]
d 25=-32.5 (c=0.90; CHCl
3)
1H?NMR[(600MHz,CDCl
3):δ=2.67(d,2H,-CH
2),2.83(d,3H,-NCH
3),5.36(t,1H,-CH),5.75(br,1H,-NH),6.96(m,2H,aromatic),7.24(m,1H,aromatic)]。
In other embodiment, substrate is identical with this example with product data.
Therefore, carbonyl reductase ChKRED15 has the function of high-selectivity reduction carbonyl, is a kind of carbonyl reductase.
Embodiment 5 HPLC detect
HPLC detects instrument: Shimadzu Prominence LC-20AD system-PDA detector.
While detecting transformation efficiency, chromatographic column is Inertsil SIL-100A250 × 4.6mm, moving phase: normal hexane: Virahol=70:30, flow velocity 1mL/min, 35 ℃ of column temperatures, the residence time of N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide and N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide is respectively 8.921min and 13.022min.
While detecting ee value, chiral chromatographic column is Daicel Chiralcel OJ-H250 × 4.6mm, moving phase: normal hexane: Virahol=90:10, flow velocity 0.5mL/min, the residence time of (S)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide and (R)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide is respectively 22.808min and 24.176min.
The pure enzymes biocatalysis of embodiment 6 carbonyl reductase ChKRED15
The pure enzyme of carbonyl reductase ChKRED15 that separation and purification in embodiment 3 is obtained is for catalytic substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide.
Reaction system 10mL, comprises potassium phosphate buffer (0.1M, pH7.0), pure enzyme concn 0.1g/L, substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 1g/L, NADH10mM.30 ℃, rotating speed 100rpm, transforms 30min.Reaction finishes rear by the long-pending ethyl acetate extracting twice of monoploid, merge organic layer and use anhydrous sodium sulfate drying, nitrogen blows down and goes ethyl acetate, and gained resistates fully dissolves with 0.1mL Virahol (HPLC level), get 2 μ l samples, according to the condition of embodiment 5, carry out HPLC detection.Calculating transformation efficiency is 28%, ee value >99.9%.
Embodiment 7 carbonyl reductase ChKRED15 coenzyme Preferences
Reaction system 10mL, comprises potassium phosphate buffer (0.1M, pH7.0), pure enzyme concn 0.1g/L, 10mM substrate and 5mM NADH or NADPH.30 ℃, rotating speed 150rpm, reaction 1h.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures the transformation efficiency of product.During take NADH as coenzyme, transformation efficiency is 17%; During take NADPH as coenzyme, transformation efficiency is 49%; Both ee values are all greater than 99.9%, and when therefore this enzyme is take NADPH as coenzyme, transformation efficiency is higher.
The bioreduction of embodiment 8 carbonyl reductase ChKRED15 under differing temps
The biocatalysis temperature of choosing is respectively 5 ℃, 10 ℃, 15 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 40 ℃.
Reaction system 10mL, comprises potassium phosphate buffer (0.1M, pH7.0), the pure enzyme concn 0.1g/L of carbonyl reductase ChKRED15, N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 10mM, 10mM NADPH, respectively under said temperature, react 1h, rotating speed 200rpm.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product.The results are shown in Table 1.
The bioreduction transformation efficiency of table 1 carbonyl reductase ChKRED15 under differing temps
Result shows, carbonyl reductase ChKRED15 all can conversion of substrate within the scope of 5 ℃～40 ℃, at 24 ℃～32 ℃ transformation efficiencys, is more or less the same.Under above temperature condition, product ee value is all greater than 99.9%.
Embodiment 9 carbonyl reductase ChKRED15 are at the bioreduction of different pH values
Buffer system comprises: potassium phosphate buffer (pH6.0～8.0,0.1M), Tris – HCl (pH8.0 – 9.0,0.1M).Transformation system 10mL, except damping fluid, other components are: pure enzyme concn 0.1g/L, N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 10mM and 10mM NADPH.Temperature of reaction is 28 ℃, rotating speed 100rpm, and the reaction times is 1h.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product.The results are shown in Table 2.
Table 2 carbonyl reductase ChKRED15 is at the bioreduction of different damping fluids and pH value
Result shows that carbonyl reductase ChKRED15 all can carry out bioreduction in two kinds of damping fluids, and pH scope is 6.0～9.0 left and right.During potassium phosphate buffer pH8.0, show the highest transformation efficiency.The product ee value of above catalyzed reaction is all greater than 99.9%.
The suitable buffer concentration of embodiment 10 carbonyl reductase ChKRED15 biocatalysis
Take pH8.0 potassium phosphate buffer as buffer system, buffer concentration is respectively: 0.1M, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, 2.2M, 2.4M.
Transformation system 10mL, except damping fluid, other components are: pure enzyme concn 0.1g/L, N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 10mM and 10mM NADPH.Temperature of reaction is 28 ℃, rotating speed 120rpm, and the reaction times is 30min.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product.The results are shown in Table 3.
The biocatalysis of table 3 carbonyl reductase ChKRED15 under different buffer concentrations
Result shows that carbonyl reductase ChKRED15 is in pH8.0 potassium phosphate buffer, and buffer concentration is that 0.1～2.4M left and right all can transform target substrates.When buffer concentration is 1.0～1.4M, transformation efficiency is more or less the same.The product ee value of above catalyzed reaction is all greater than 99.9%.
Embodiment 11 carbonyl reductase ChKRED15 recombinant bacterium whole-cell biological catalysis
Carbonyl reductase ChKRED15 recombinant bacterium E.coli(pET28a-ChKRED15) cultural method and microorganism collection method see embodiment 2.
Get thalline 1.0g, with potassium phosphate buffer (pH8.0,0.3M), being made into cell concn is 100g/l bacteria suspension.5mL reaction system, substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide concentration is 10g/L, add 10%(w/v) glucose as regenerating coenzyme substrate, 28 ℃, 230rpm, transform 24h, the long-pending ethyl acetate extracting twice of monoploid for reaction mixture, merge organic layer and use anhydrous sodium sulfate drying, underpressure distillation will be removed ethyl acetate, gained resistates fully dissolves with 2mL Virahol (HPLC level), get 2 μ L samples and carry out HPLC detection, sample treatment is identical with embodiment 6, calculating transformation efficiency is >99%, ee value >99.9%.
Embodiment 12 carbonyl reductase ChKRED15 Cofactor Regeneration Systems
Carbonyl reductase ChKRED15 recombinant bacterium E.coli(pET28a-ChKRED15) derivational expression method see embodiment 2, separation and purification of protein method is shown in embodiment 3.
(1) GDH concentration optimization in carbonyl reductase ChKRED15 regenerating coenzyme circulating system
Transformation system 10mL: potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED15 concentration transforms the required enzyme amount of 1 μ mol substrate for 2U/mL(1 minute and is defined as 1U), substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 10mM, NADP
+0.2mM, glucose 10%, Hexose phosphate dehydrogenase (GDH) concentration is respectively 0U/mL, 0.1U/mL, 0.2U/mL, 0.5U/mL, 1U/mL, 2U/mL, 4U/mL, 6U/mL, 28 ℃, 150rpm, transforms 60min.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product, and optimum result is in Table 4.
GDH concentration optimization in table 4 carbonyl reductase ChKRED15 regenerating coenzyme circulating system
Result shows when GDH concentration is 2U/mL, and regenerating coenzyme circulating system can meet the demand of carbonyl reductase ChKRED15 to NADPH substantially.Above catalytic reaction products ee value is all greater than 99.9%.
(2) NADP in carbonyl reductase ChKRED15 regenerating coenzyme circulating system
+concentration optimization
Transformation system 10mL: potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED15 concentration transforms the required enzyme amount of 1 μ mol substrate for 2U/mL(1 minute and is defined as 1U), substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 110mM, GDH concentration 2U/mL, glucose 10%, NADP
+concentration is respectively 0mM, 0.01mM, and 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 28 ℃, 100rpm transforms 60min.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product, and optimum result is in Table 5.
NADP in table 5 carbonyl reductase ChKRED15 regenerating coenzyme circulating system
+concentration optimization
Result shows that the transformation efficiency of carbonyl reductase ChKRED15 catalytic substrate is with NADP
+concentration increases and increases, and works as NADP
+when concentration is greater than 0.5mM, transformation efficiency tends towards stability.Above catalytic reaction products ee value is all greater than 99.9%.
Embodiment 13 carbonyl reductase ChKRED15 crude enzyme liquid biocatalysis
The derivational expression method of carbonyl reductase ChKRED15 recombinant bacterium is shown in embodiment 2.
The wet thallus of getting fresh culture is resuspended in phosphate buffered saline buffer (0.1M, pH8.0), with clarifixator smudge cells, and the centrifugal 20min of 13 000rpm, gained supernatant is crude enzyme liquid.
Transformation system 10mL: potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED15 concentration transforms the required enzyme amount of 1 μ mol substrate for 2U/mL(1 minute and is defined as 1U), substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 20g/L, GDH concentration 2U/mL, glucose 10%(w/v), NADP
+concentration 0.2mM, 28 ℃, rotating speed 150rpm, transformation time is respectively 10min, 20min, 30min, 40min, 50min, 60min, 90min, 120min.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product, the results are shown in Table 6.
Table 6 carbonyl reductase ChKRED15 crude enzyme liquid biocatalysis
Result shows that in 10min, substrate just can transform 15% left and right, and 120min substrate transforms completely, and the ee value of product is greater than 99.9%.
The thick enzyme powder of embodiment 14 carbonyl reductase ChKRED15 biocatalysis
The method that obtains carbonyl reductase ChKRED15 crude enzyme liquid is identical with embodiment 13; Crude enzyme liquid is frozen in to-80 ℃ and spends the night, then with frozen vacuum dryer, be dried, make thick enzyme powder.Measure the enzyme (transform the required enzyme amount of 1 μ mol substrate in 1 minute and be defined as 1U) alive of thick enzyme powder.
Transformation system 10mL: potassium phosphate buffer (0.1M, pH8.0), carbonyl reductase ChKRED15 concentration 2U/mL, substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide 20g/L, GDH concentration 2U/mL, glucose 10%(w/v), NADP
+concentration 0.2mM, 28 ℃, rotating speed 160rpm, transformation time is respectively 10min, 20min, 30min, 40min, 50min, 60min, 90min, 120min.Equal-volume ethyl acetate extracting twice, sample treatment is identical with embodiment 6, measures transformation efficiency and the ee value of product, the results are shown in Table 7.
The thick enzyme powder of table 7 carbonyl reductase ChKRED15 biocatalysis
Result shows that in 10min, substrate just can transform 14% left and right, and 120min substrate transforms completely, and the ee value of product is greater than 99.9%.
Claims (3)
1. a carbonyl reductase
chthe gene of KRED15, its nucleotides sequence is classified as shown in SEQ ID No.1.
2. a carbonyl reductase
chkRED15, its aminoacid sequence is shown in SEQ ID No.2.
3. carbonyl reductase claimed in claim 2
chkRED15 as biological catalyst conversion of substrate N-methyl-3-oxygen-3-(2-thienyl) propionic acid amide generate (
sapplication in)-N-methyl-3-hydroxyl-3-(2-thienyl) propionic acid amide.
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| CN104830924A (en) * | 2015-05-20 | 2015-08-12 | 中国科学院成都生物研究所 | Method for producing thienyl chiral alcohol compound through biological catalysis of carbonyl reductase |
| CN105803013A (en) * | 2016-06-02 | 2016-07-27 | 江南大学 | Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase |
| CN106520855A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Method for preparing stereoscopic complementary N-heterocycle alcohol compounds by conducting biological catalysis through carbonyl reductase |
| CN110229796A (en) * | 2019-05-23 | 2019-09-13 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN112852767A (en) * | 2019-12-06 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in catalytic synthesis of 17 beta-hydroxy steroid compound |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013898A (en) * | 2012-11-07 | 2013-04-03 | 宁波美诺华药业股份有限公司 | Carbonyl reductase expressed recombination engineering bacterium and application thereof |
| CN103497911A (en) * | 2013-09-05 | 2014-01-08 | 中国科学院成都生物研究所 | Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate |
-
2014
- 2014-01-12 CN CN201410012492.6A patent/CN103740738B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013898A (en) * | 2012-11-07 | 2013-04-03 | 宁波美诺华药业股份有限公司 | Carbonyl reductase expressed recombination engineering bacterium and application thereof |
| CN103497911A (en) * | 2013-09-05 | 2014-01-08 | 中国科学院成都生物研究所 | Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate |
Non-Patent Citations (1)
| Title |
|---|
| 应国清，等: "马克斯克鲁维酵母羰基还原酶基因的克隆与表达", 《微生物学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830924A (en) * | 2015-05-20 | 2015-08-12 | 中国科学院成都生物研究所 | Method for producing thienyl chiral alcohol compound through biological catalysis of carbonyl reductase |
| CN104830924B (en) * | 2015-05-20 | 2018-04-27 | 中国科学院成都生物研究所 | Carbonyl reduction enzymes biocatalysis produces thienyl chirality alcohol compound |
| CN105803013A (en) * | 2016-06-02 | 2016-07-27 | 江南大学 | Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase |
| CN105803013B (en) * | 2016-06-02 | 2019-05-10 | 江南大学 | A method of utilizing carbonyl reductase asymmetric syntheses duloxetine intermediate |
| CN106520855A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Method for preparing stereoscopic complementary N-heterocycle alcohol compounds by conducting biological catalysis through carbonyl reductase |
| CN110229796A (en) * | 2019-05-23 | 2019-09-13 | 南京趣酶生物科技有限公司 | Ketoreductase mutant and its preparing the application in Duloxetine chirality alcohol intermediate and the like |
| CN110229796B (en) * | 2019-05-23 | 2023-09-05 | 安徽趣酶生物科技有限公司 | Ketoreductase mutant and application thereof in preparation of duloxetine chiral alcohol intermediate and analogue thereof |
| CN112852767A (en) * | 2019-12-06 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in catalytic synthesis of 17 beta-hydroxy steroid compound |
| CN112852767B (en) * | 2019-12-06 | 2022-04-05 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in catalytic synthesis of 17 beta-hydroxy steroid compound |
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