CN104630171A - New (R)-transaminase from Fusarium oxysporum and application thereof - Google Patents
New (R)-transaminase from Fusarium oxysporum and application thereof Download PDFInfo
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Abstract
The invention discloses a new (R)-transaminase and application thereof. The (R)-transaminase derives from Fusarium oxysporum Fo5176 (named HFO), the gene nucleotide sequence is shown as SEQ ID No.1 and the amino acid sequence is shown as SEQ ID No.2. The new (R)-transaminase HFO is a strict (R) stereoselective Omega-transaminase, has a broad substrate spectrum, and has great application potential in bio-production of chiral amines and unnatural amino acids.
Description
Technical field
The present invention relates to biocatalysis field, relate generally to and a kind ofly derive from new (the R)-transaminase of Fusarium oxysporum and the application in production Chiral Amine and alpha-non-natural amino acid thereof.
Background technology
Chiral Amine and alpha-non-natural amino acid have a wide range of applications at value segments such as medicine, food, makeup.Such as (R)-4-benzene butyl-2-amine (precursor of antihypertensive drug Dilevalol), phenylisoserine (beta-amino acids, the pendant moiety of anticarcinogen taxol) and Cispentacin (ring-type beta-amino acids, antifungal drug) etc. be all the key intermediate of pharmaceutical synthesis or crucial handedness module, the alpha-non-natural amino acids such as D-alanine are more and more is used to superior cosmetics.In addition, research shows, the stability of the polypeptide reply proteasome degradation containing alpha-non-natural amino acid higher than natural polypeptides, and still keeps its biological activity.This is presented at research and development high stability and not by new drug, antibody and artificial protein aspect that human body repels, alpha-non-natural amino acid maybe will play irreplaceable effect.But different from natural amino acid, Chiral Amine and alpha-non-natural amino acid still do not carry out suitability for industrialized production by fermentation method, the main organic synthesis method that relies on is produced now.The deficiency of organic synthesis method is difficult to obtain optically pure product, and usually need high pressure-temperature high energy consumption, therefore needs the green production realizing Chiral Amine and alpha-non-natural amino acid badly.
Transaminase, is transaminase again, is the class of enzymes of amino reversible transfer (ping-pong mechanism) between catalytic amino compound and carbonyl compound, according to the position of its substrate amino, can be divided into α-transaminase and ω-transaminase.α-transaminase is comparatively common, can only the alpha-amino transfer of catalysis; ω-transaminase is then comparatively rare, except the alpha-amino transfer of energy catalysis, also can the transfer of catalysis non alpha position amino, have more broad substrate spectrum and strict stereoselectivity, the asymmetric ammonification (accompanying drawing 1b) that can split in a mild condition on amine raceme (accompanying drawing 1a) or catalysis prochiral substrate carbonyl comes production Chiral Amine and alpha-non-natural amino acid.In addition, ω-transaminase is with pyridoxal phosphate (PLP) for prothetic group, and without the need to adding extra cofactor circulation in catalytic reaction process, this is advantageous in industrial application.In a word, produce optically pure Chiral Amine and alpha-non-natural amino acid by ω-transaminase, the high pressure-temperature high energy consumption high pollution of conventional chemical methods can be avoided, be that the advanced person's biology meeting green production theory manufactures, demonstrate the great potential of alternative organic synthesis method.Such as, within 2010, Science reports the achievement in research of Codexis company and Merck company, adopts (R) chemical method that optionally ω-transaminase successfully instead of based on Noble Metal Rhodium catalysis to carry out suitability for industrialized production antidiabetic medicine sitagliptin (a kind of Chiral Amine).
Strict stereoselectivity is the key characteristic that enzyme is different from chemical catalyst, so obtain specific stereoselective enzyme, is the important foundation of follow-up study transformation and industrial application.According to bibliographical information, in ω-transaminase (S) optionally enzyme is comparatively common, and (R) optionally comparatively rare ([1] Koszelewski D of ω-transaminase (hereinafter referred to as (R)-transaminase), Tauber K, Faber K, et al. ω-Transaminases for the synthesis of non-racemic α-chiral primary amines [J] .Trends in biotechnology, 2010, 28 (6): 324-332. [2] Mathew S, Yun H. ω-Transaminases for the production of optically pure amines and unnatural amino acids [J] .Acs Catalysis, 2012, 2 (6): 993-1001).Up to now, the domestic report not yet having (R)-transaminase.Therefore, the green production of excavating the chiral amine of new (R)-transaminase and alpha-non-natural amino acid with industrial application potentiality has special meaning.
Summary of the invention
The green production that the object of the invention is for Chiral Amine and alpha-non-natural amino acid provides a kind of (R)-transaminase with industrial application potentiality.
The present invention seeks to be achieved through the following technical solutions:
A kind of new (R)-transaminase (being called HFO) deriving from Fusarium oxysporum Fusarium oxysporum Fo5176, its gene nucleotide series is as shown in SEQ NO.1, and its aminoacid sequence is as shown in SEQ NO.2.
This area investigator carries out to new (R)-transaminase HFO (R)-transaminase that protein engineering transformation (such as amino-acid residue conservative substitution, aminoacid sequence increase the chemically modified etc. of brachymemma, appropriate design, orthogenesis, enzyme) obtains, and also belongs to scope of the present invention.
Another object of the present invention is to provide the method that one prepares newly (R)-transaminase HFO.
This preparation method comprises the following steps: (1) obtains the gene fragment of new (the R)-transaminase HFO of coding by round pcr.(2) gene fragment is building up on pET28a expression vector, obtains the recombinant plasmid with object enzyme gene.(3) recombinant plasmid is proceeded to Host Strains cell (preferred e. coli bl21 (DE3)), obtain corresponding engineering strain.(4) engineering strain is seeded in LB substratum, is cultured to logarithmic phase and with the IPTG of 0.1mM in 25 DEG C of abduction deliverings 12 hours.(5) collected by centrifugation thalline, broken bacterium leaves and takes supernatant liquor, adopts metal affinity chromatography method (nickel post) purifying to reclaim target protein, and namely dialysis removing imidazoles obtains the pure enzyme liquid of HFO.SDS-PAGE electrophoretogram display target protein new (R)-transaminase HFO major part soluble-expression, purifying gained protein band is single, reaches electrophoresis pure (see accompanying drawing 2).
Another object of the present invention is to provide zymologic property, the substrate spectrum of newly (R)-transaminase HFO and applies.
New (R)-transaminase HFO vigor under weak basic condition of the present invention is higher, and its optimal reaction pH value is 8.0.Newly the optimal reactive temperature of (R)-transaminase HFO is 35 DEG C, and it still keeps the vigor of more than 90% hatch 20 minutes in the phosphate buffered saline buffer (100mM, pH=7.4) of 45 DEG C after.
New (R)-transaminase HFO of the present invention has broad amino group donor substrate spectrum, especially very high to the vigor of various (R) configuration primary amine, such as (R)-1-phenylethylamine, (R)-2-hexylamine and (R)-2-heptyl amice, and very low to the vigor of (S) configuration primary amine, show strict (R) stereoselectivity.New (the R)-transaminase HFO of this display can be used for the kinetic resolution of primary amine raceme.
Further, new (R)-transaminase HFO of the present invention can the 1-phenylethylamine of kinetic resolution 50mM, 1-amphetamine, 1-aminoidan and 1-(4-fluorophenyl) ethamine, transformation efficiency is all about 50%, ee value all >99% of product, obtain optically pure (S)-1-phenylethylamine (accompanying drawing 3), (S)-1-amphetamine (accompanying drawing 4), (S)-aminoidan (accompanying drawing 5) and (S)-(4-fluorophenyl) ethamine (accompanying drawing 6).These results, fully show the application potential of new (R)-transaminase HFO of the present invention in kinetic resolution amine raceme acquisition Chiral Amine.
New (R)-transaminase HFO of the present invention has broad amino acceptor substrate spectrum, comprise ketone, hydroxyketone, alpha-ketoacid and beta-ketoester etc., and the ee value of its corresponding product is mostly more than 99%.New (the R)-transaminase HFO of this display can be applicable to the asymmetric synthesis of Chiral Amine and alpha-non-natural amino acid.
Further, new (R)-transaminase HFO of the present invention can add ammonia synthesis D-α-alanine by asymmetric for 50mM pyruvic acid, and transformation efficiency reaches 99%, ee value >99% (accompanying drawing 7); Can add ammonia synthesis D-butyrine by asymmetric for the 2-ketone butyric acid of 20mM and 50mM, transformation efficiency reaches 99% and 92%, ee value all >99% (accompanying drawing 8) respectively; Can add ammonia synthesis D-butalanine by asymmetric for the 2-ketone valeric acid of 10mM, transformation efficiency reaches 85%, ee value >99% (accompanying drawing 9).These results, fully show new (R)-transaminase HFO of the present invention and be rich in application potential in the asymmetric synthesis of optical purity alpha-non-natural amino acid.
The invention has the advantages that: (R)-transaminase is a class ω-transaminase rarer than (S)-transaminase, acquire a special sense in the biology manufacture of Chiral Amine and alpha-non-natural amino acid.New (R)-transaminase HFO that the present invention announces has strict (R) stereoselectivity, and has broad substrate spectrum, in the green production of Chiral Amine and alpha-non-natural amino acid, demonstrate prospects for commercial application.
Accompanying drawing explanation
Accompanying drawing 1 is ω-transaminase-catalyzed reaction schematic diagram.
Accompanying drawing 2 is the SDS-PAGE collection of illustrative plates of new (R)-transaminase HFO.1: molecular weight protein marker, 2: ni-sepharose purification gained HFO, 3: bacteria break supernatant liquid after abduction delivering, 4: broken bacterium precipitation after abduction delivering.
Accompanying drawing 3 is that the product optical purity of HFO kinetic resolution 1-phenylethylamine detects collection of illustrative plates.
Accompanying drawing 4 is that the product optical purity of HFO kinetic resolution 1-amphetamine detects collection of illustrative plates.
Accompanying drawing 5 is that the product optical purity of HFO kinetic resolution 1-aminoidan detects collection of illustrative plates.
Accompanying drawing 6 is that the product optical purity of HFO kinetic resolution 1-(4-fluorophenyl) ethamine detects collection of illustrative plates.
Accompanying drawing 7 is that the product optical purity of the asymmetric ammonification of HFO catalysis pyruvic acid detects collection of illustrative plates.
Accompanying drawing 8 is that the product optical purity of the asymmetric ammonification of HFO catalysis 2-butanone acid detects collection of illustrative plates.
Accompanying drawing 9 is that the product optical purity of the asymmetric ammonification of HFO catalysis 2 pentanone acid detects collection of illustrative plates.
Embodiment
The following examples further illustrate partial content of the present invention, so that those skilled in the art understands the present invention further, but should not be construed as limitation of the present invention.
The preparation method of new (the R)-transaminase HFO of embodiment one
(1) engineering bacteria is built: the encoding gene (SEQ NO.1) of new (R)-transaminase HFO increase by round pcr and obtains from Fusarium oxysporum Fusarium oxysporum Fo5176 genomic dna (N holds and is added with 6 × His-tag to purify target protein HFO with nickel affinity column), and upstream primer is: catg
cCATGGcTCACCATCATCATCATCACATGGCAACTATGGACGAGG, downstream primer is ccc
aAGCTTcTACTAGTAATCAATCTTGAAGCTGAACTC, institute's PCR fragment that obtains inserts pET28a expression vector in Nco I/Hind III site after double digestion.Recombinant plasmid is transferred into Host Strains cell (preferred e. coli bl21 (DE3)), obtains corresponding engineering strain.
(2) expression and purification: adopt LB substratum, be cultured to logarithmic phase in 37 DEG C of shaking tables, after being cooled to 25 DEG C, add the IPTG that final concentration is 0.1mm, abduction delivering 12 hours in 25 DEG C of shaking tables.4 DEG C, collected by centrifugation thalline under 6000rpm condition, with the sodium phosphate buffer (50mM containing 20 μMs of pyridoxal phosphates (PLP), pH7.0) clean twice, and use high-pressure homogenization crusher machine, 13000rpm is centrifugal leaves and takes supernatant liquor, then adopts metal affinity chromatography (nickel post) method purifying to reclaim target protein, and namely target protein obtains newly the pure enzyme liquid of (R)-transaminase HFO after dialysis removing imidazoles.
New (the R)-transaminase HFO major part of SDS-PAGE electrophoresis showed target protein is present in cell with soluble form, the purity of gained target protein very high (see accompanying drawing 2) after ni-sepharose purification.
The zymologic property of new (the R)-transaminase HFO of embodiment two
Examine the optimum pH of new (R)-transaminase HFO, optimal reactive temperature and temperature stability.
Reaction skeleton symbol:
.
Survey the pH damping fluid of reaction system: the 1ml that lives, PLP (20 μMs), (R)-1-phenylethylamine (10mM), pyruvic acid (10mM).Start reaction by adding appropriate new (R)-transaminase HFO, reaction half an hour after with perchloric acid termination reaction.After termination reaction, the amount of the methyl phenyl ketone generated by HPLC assaying reaction determines the size of enzyme activity.
The mensuration of optimal reaction pH value: adopt and survey reaction system of living, temperature of reaction 30 DEG C, is determined at the enzyme activity size of a series of different pH value (pH=5,6,7,7.4,7.8,8,8.4,8.8,9,9.4,9.6,10) stylish (R)-transaminase HFO.Different pH scope damping fluid used is: acetate buffer solution (pH3.8-5.6), sodium phosphate buffer (pH5.8-7.6), boric acid borate buffer solution (pH7.8-9.2) and boraxsodium hydroxide buffer (pH9.3-10.1).
The mensuration of optimal reactive temperature: adopt and survey reaction system of living, select sodium phosphate buffer (100mM, pH7.4), the enzyme activity size of new (R)-transaminase HFO under being determined at a series of differential responses temperature (25,30,35,37,40,42,45,50 DEG C).
Temperature stability: adopt and survey reaction system of living, select sodium phosphate buffer (100mM, pH7.4), temperature of reaction is 30 DEG C, measures the size of the residual enzyme vigor respectively through a series of differing temps (30,35,40,45,50,55,60 DEG C) thermal treatment new (the R)-transaminase HFO of 20 minutes.
Result shows: new (R)-transaminase HFO vigor under weak basic condition is higher, and its optimal reaction pH value is 8.0; Optimal reactive temperature is 35 DEG C, and it still keeps the vigor of more than 90% hatch 20 minutes in the phosphate buffered saline buffer (100mM, pH7.4) of 45 DEG C after.
The substrate spectrum of new (the R)-transaminase HFO of embodiment three and application
(1) the amino group donor substrate spectrum of new (R)-transaminase HFO.
Reaction skeleton symbol: amino group donor (table 3)
Reaction conditions: reaction system 1ml.At sodium phosphate buffer (100mM, pH7.4) pyridoxal phosphate (20 μMs) is added in, amino group donor substrate (10mM, raceme 20mM) and pyruvic acid (10mM), add appropriate HFO enzyme liquid after mixing and start reaction, react in 30 DEG C of water-baths after 30 minutes, by the 16% perchloric acid termination reaction of 375ul.
The calculating of relative activity: calculate the relative activity of each substrate by the reduction of pyruvic acid in detection reaction system.The Liquid Detection condition of pyruvic acid is: Aminex HPX-87H post (Bio-Rad, USA), and column temperature 45 DEG C, with the H of 5mM
2sO
4for moving phase, flow velocity 0.6ml/min, detects the ultraviolet absorption value at 205nm place.
Result is as shown in table 3, newly the vigor of (R)-transaminase HFO to different amino group donor is different, it is very high to the vigor of amine, and to the vigor of aliphatic amide higher than the vigor to aromatic amine, (R)-2-hexylamine and (R)-2-heptyl amice are two kinds of amino group donor that vigor is the highest.Newly the vigor of (R)-transaminase HFO to (R)-1-phenylethylamine is very high, and very low to (S)-1-phenylethylamine vigor, illustrates that this enzyme has strict (R)-stereoselectivity.Result shows: new (R)-transaminase HFO obtains in (S) configuration Chiral Amine at fractionation amine raceme has application potential.
The amino group donor substrate spectrum of new (the R)-transaminase HFO of table 3
Table note: 1. the vigor of enzyme to (R)-1-phenylethylamine (10mM) is decided to be 100% (8.8U/mg), relative activity is less than 1% and is designated as zero.2. a Ge Meihuo unit (1U) is defined as in the reaction system being amino group donor and amino acceptor with (R)-1-phenylethylamine (10mM) and pyruvic acid (10mM) respectively, and in 1 minute, catalysis forms the enzyme amount required for 1 μm of ol methyl phenyl ketone.
(2) the amino acceptor substrate spectrum of new (R)-transaminase HFO.
Reaction skeleton symbol: (R)-1-phenylethylamine+amino acceptor (table 4)
Reaction conditions: reaction system 1ml.At sodium phosphate buffer (100mM, pH7.4) pyridoxal phosphate (20 μMs) is added in, (R)-1-phenylethylamine (10mM) and amino acceptor substrate (10mM), add appropriate HFO enzyme liquid after mixing and start reaction, react in 30 DEG C of water-baths after 30 minutes, by the 16% perchloric acid termination reaction of 375ul.
The calculating of relative activity: calculate the relative activity of each substrate by the growing amount of methyl phenyl ketone in detection reaction system.The testing conditions of methyl phenyl ketone is: Eclipse XDB-C18 post (4.6 × 150mm, Aglient, USA), acetonitrile/water (50/50, v/v) isocratic elution, flow velocity 1ml/min, detects the ultraviolet absorption value at 205nm place.
The detection of product configuration: the ammonification product beta-aminobutyric acid ethyl ester of (1) substrate acetyl ethyl acetate and Propionylacetic acid ethyl ester and beta-amino Valeric acid ethylester: unreacted substrate is removed in the extracting of reaction solution furnishing slightly acidic ethyl acetate, aqueous phase is adjusted to weakly alkaline, extract product by ethyl acetate, in stink cupboard, ethyl acetate vapored away and obtain product.Carry out acetylize to product to derive, use gas chromatographic detection afterwards.Testing conditions is: Chirasil-DEX CB post (25m × 0.25mm × 0.25 μm, Varian, USA), column temperature 140 DEG C, constant temperature, splitting ratio 20:1, carrier gas (helium) flow velocity 2ml/min, fid detector 220 DEG C.(2) the ammonification product that in table 5, other substrates are corresponding: get appropriate reaction solution, FDAA (1-fluoro-2 is carried out to it, 4-dinitrophenyl-5-L-alanimamides) derivative, operation is with reference to specification sheets (Thermo Fisher Scientific, USA).Use liquid chromatographic detection afterwards.Testing conditions is: Eclipse XDB-C18 post, phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile (50/50, v/v) isocratic elution, detects the ultraviolet absorption peak at 340nm place.To different product, phosphoric acid triethylamine (50mM, the ratio of pH3.0)/acetonitrile is different with flow velocity: to the product of pyruvic acid, 2-butanone acid, sour, the 4-oxopentanoic acid of 2 pentanone and pyruvic alcohol, phosphoric acid triethylamine (50mM, the ratio of pH3.0)/acetonitrile is 70/30 (v/v), flow velocity 0.5ml/min; To the product of 4-Phenyl 2 butanone, 2 pentanone and methyl-n-butyl ketone, the ratio of phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile is 50/50 (v/v), flow velocity 1ml/min; To the product of mercaptopyruvate, the ratio of phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile is 70/30 (v/v), flow velocity 1ml/min.
Result is as shown in table 4, and new (R)-transaminase HFO shows obvious vigor to all receptor substrates tested, and the optical purity of corresponding product is very high, most ee value >99% (R).Pyruvic acid is the amino acceptor substrate that vigor is the highest, and the vigor of 2 pentanone acid and oxoethanoic acid is also very high, and new (R)-transaminase HFO also shows obvious vigor to ketone substrate.Especially meaningfully, newly (R)-transaminase HFO has remarkable vigor to pyruvic acid, 2-butanone acid, 2 pentanone sour, mercaptopyruvate, methyl aceto acetate, Propionylacetic acid ethyl ester and 4-oxopentanoic acid, the ammonification product that these substrates are corresponding is all alpha-non-natural amino acid (or its esterified form), and new (the R)-transaminase HFO of this display has application potential in asymmetric synthesis alpha-non-natural amino acid.
The amino acceptor substrate spectrum of new (the R)-transaminase HFO of table 4
Table note: 1. the vigor of enzyme to pyruvic acid (10mM) is decided to be 100% (8.8U/mg), relative activity is less than 1% and is designated as zero.2. a Ge Meihuo unit (1U) is defined as in the reaction system being amino group donor and amino acceptor with (R)-1-phenylethylamine (10mM) and pyruvic acid (10mM) respectively, and in 1 minute, catalysis forms the enzyme amount required for 1 μm of ol methyl phenyl ketone.
(3) new (R)-transaminase HFO kinetic resolution amine raceme.
Kinetic resolution reaction schematic diagram: accompanying drawing 1a.
Reaction conditions: reaction system is 1ml, amino group donor is racemic amine (1-phenylethylamine, 1-amphetamine, 1-aminoidan or 1-(4-fluorophenyl) ethamine), and amino acceptor is pyruvic acid.In sodium phosphate buffer (100mM, pH7.4), add the pyridoxal phosphate of 0.1mM, the pyruvic acid of 40mM, the racemic amine of 50mM, new (the R)-transaminase HFO of 1.2mg, be placed in 30 DEG C of shaking table reactions 15 hours.Reaction arranges blank (not enzyme-added), to calculate transformation efficiency.
The calculating of transformation efficiency: transformation efficiency calculates according to the reduction of primary amine in reaction system, namely in transformation efficiency=reaction system primary amine reduction ÷ blank in total amount × 100% of primary amine.The testing conditions of primary amine content is: Eclipse XDB-C18 post (4.6 × 150mm, Aglient, USA), acetonitrile/water (50/50, v/v) isocratic elution, flow velocity 1ml/min, detects the ultraviolet absorption value at 205nm place.
The detection of product amine optical purity: get appropriate reaction solution, the protein of first centrifuging removing sex change, afterwards FDAA (1-fluoro-2 is carried out to it, 4-dinitrophenyl-5-L-alanimamides) derivative, operation is with reference to specification sheets (Thermo Fisher Scientific, USA).Derived products liquid chromatographic detection, testing conditions: Eclipse XDB-C18 post, phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile (50/50, v/v) isocratic elution, flow velocity 1ml/min, detects the ultraviolet absorption value at 340nm place.Relative content according to the corresponding configuration of product calculates product ee value.
Result is as shown in table 5, newly the transformation efficiency of (R)-transaminase HFO kinetic resolution 50mM racemic amine is all about 50%, product ee value is all greater than 99% (S) (accompanying drawing 3, accompanying drawing 4, accompanying drawing 5, accompanying drawing 6), illustrate that (R) configuration amine is almost fully used, obtain optically pure (S) configuration amine.Result vivo shows: new (R)-transaminase HFO catalytic condition of the present invention is gentle, reaction conversion ratio is good, product optical purity is high, has very large using value in the green production of Chiral Amine.
New (the R)-transaminase HFO kinetic resolution amine raceme of table 5
(4) new (R)-transaminase HFO catalyzes and synthesizes alpha-non-natural amino acid.
Asymmetric catalysis synthesis schematic diagram: accompanying drawing 1b.
Reaction conditions: reaction system is 1ml, with racemization 1-phenylethylamine for amino group donor, with pyruvic acid, 2-butanone acid and the sour three kinds of alpha-ketoacids of 2 pentanone for amino acceptor.At sodium phosphate buffer (100mM, pH7.4) in, add the pyridoxal phosphate of 0.1mM, a kind of substrate alpha-ketoacid (10mM, 20mM or 50mM), the racemization 1-phenylethylamine (30mM, 60mM or 50mM) of three times of equivalents, newly (R)-transaminase HFO, is placed in 30 DEG C of shaking tables and reacts.Reaction arranges blank (not enzyme-added), to calculate transformation efficiency.
Further, when substrate alpha-ketoacid concentration is 10mM or 20mM, the consumption of new (R)-transaminase HFO is 0.8mg/ml, and the reaction times is 12 hours; When substrate alpha-ketoacid concentration is 50mM, the consumption of new (R)-transaminase HFO is 1.2mg/ml, and the reaction times is 16 hours.
The calculating of transformation efficiency: transformation efficiency calculates according to the reduction of substrate alpha-ketoacid in reaction system, namely in transformation efficiency=reaction system substrate alpha-ketoacid reduction ÷ blank in total amount × 100% of substrate alpha-ketoacid.The testing conditions of substrate alpha-ketoacid content is: Aminex HPX-87H post (Bio-Rad, USA), and column temperature 45 DEG C, with the H of 5mM
2sO
4for moving phase, flow velocity 0.6ml/min, detects the ultraviolet absorption value at 205nm place.
The detection of product alpha-non-natural amino acid configuration: get appropriate reaction solution, the protein of first centrifuging removing sex change, afterwards FDAA (1-fluoro-2 is carried out to it, 4-dinitrophenyl-5-L-alanimamides) derivative, operation is with reference to specification sheets (Thermo Fisher Sciemific, USA).Derived products liquid chromatographic detection, testing conditions: Eclipse XDB-C18 post, moving phase is phosphoric acid triethylamine (50mM, pH3.0)/acetonitrile=70/30 (v/v), flow velocity 0.5ml/min, isocratic elution, detects the ultraviolet absorption value at 340nm place.Relative content according to the corresponding configuration of product calculates product ee value.
Result is as shown in table 6, and the transformation efficiency of new (R)-transaminase HFO catalysis 50mM pyruvic acid reaches 99%, and product ee value is greater than 99% (accompanying drawing 7); The transformation efficiency of the 2-butanone acid of newly (R)-transaminase HFO catalysis 20mM and 50mM reaches 99% and 92% respectively, and product ee value is all greater than 99% (accompanying drawing 8); The transformation efficiency of the 2 pentanone acid of newly (R)-transaminase HFO catalysis 10mM reaches 85%, and product ee value is greater than 99% (accompanying drawing 9).Result vivo shows: new (R)-transaminase HFO catalytic condition of the present invention is gentle, reaction conversion ratio is good, product optical purity is high, has very large using value in the green production of alpha-non-natural amino acid.
New (the R)-transaminase HFO of table 6 catalyzes and synthesizes alpha-non-natural amino acid
Claims (10)
1. one kind derives from new (the R)-transaminase (called after HFO) of Fusarium oxysporum Fo5176, it is characterized in that: the nucleotide sequence of (1) its gene is as shown in SEQ NO.1, and its aminoacid sequence is as shown in SEQ NO.2; (2) this enzyme is expressed good under the IPTG induction of 0.1mM in 25 DEG C, and optimal reaction pH is 8.0, and optimal reactive temperature is 35 DEG C; (3) this enzyme has broad substrate spectrum and strict stereoselectivity, can be applicable to the preparation of Chiral Amine and alpha-non-natural amino acid.
2. the recombinant plasmid containing the nucleotide sequence of new (R)-transaminase HFO described in claim 1 of can encoding or expression cassette, and the engineering strain of new (R)-transaminase HFO described in claim 1 can be expressed.
3. reach 85% and above (R)-transaminase with the Amino acid sequence identity (identity) of (R)-transaminase HFO new described in claim 1.
4. the recombinant plasmid of the nucleotide sequence containing (R)-transaminase described in coding claim 3 or expression cassette, and the engineering strain of (R)-transaminase described in claim 3 can be expressed.
5. new (R)-transaminase HFO described in pair claim 1 carries out (R)-transaminase that protein engineering transformation (amino-acid residue conservative substitution, aminoacid sequence increase the chemically modified etc. of brachymemma, appropriate design, orthogenesis, enzyme) obtains.
6. the recombinant plasmid of the nucleotide sequence containing (R)-transaminase described in coding claim 5 or expression cassette, and the engineering strain of (R)-transaminase described in claim 5 can be expressed.
7. claim 1, claim 2, claim 3, claim 4, claim 5 and (R)-transaminase according to claim 6, recombinant plasmid, expression cassette or the engineering strain application in preparation Chiral Amine and alpha-non-natural amino acid.
8. apply as claimed in claim 7, it is characterized in that: when production Chiral Amine and alpha-non-natural amino acid, both comprise the Kinetic Resolution adopted amine (amino acid) raceme, also comprise the dissymmetric synthesis adopted prochiral carbonyl compounds ammonification.
9. amine (amino acid) raceme as claimed in claim 8, refers to that described in claim 1, new (R)-transaminase HFO has the racemic amine (amino acid) of catalysis activity to it, amine (amino acid) raceme that in amine (amino acid) raceme that most preferably in table 3, amino group donor substrate is corresponding and table 4, amino acceptor substrate is corresponding: 1-phenylethylamine, 1-amphetamine, 1-aminoidan, the amino tetralin of 1-, 1-(4-fluorophenyl) ethamine, 2-amylamine, 2-hexylamine, 2-heptyl amice, alpha-amino group propyl alcohol, DL-beta-aminobutyric acid, DL-α-alanine, DL-butyrine, DL-butalanine, DL-α-halfcystine, DL-beta-aminobutyric acid ethyl ester, DL-beta-amino Valeric acid ethylester, DL-gamma-amino valeric acid, 4-phenyl-2-butylamine.
10. prochiral carbonyl compounds as claimed in claim 8, refers to that described in claim 1, new (R)-transaminase HFO has the prochiral carbonyl compounds of catalysis activity to it; Amino acceptor substrate in the prochiral carbonyl compounds that most preferably in table 3, amino group donor substrate is corresponding and table 4: the acid of methyl phenyl ketone, Propiophenone, 1-indone, ALPHA-tetralone, 4-fluoro acetophenone, 2 pentanone, methyl-n-butyl ketone, 2-heptanone, pyruvic alcohol, beta-ketobutyric acid, pyruvic acid, 2-butanone, 2 pentanone are sour, mercaptopyruvate, methyl aceto acetate, Propionylacetic acid ethyl ester, 4-oxopentanoic acid, 4-Phenyl 2 butanone, 2 pentanone, methyl-n-butyl ketone.
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| CN105733958A (en) * | 2016-03-16 | 2016-07-06 | 湖南省环境保护科学研究院 | Fusarium oxysporum and application thereof to phytoremediation of heavy metal contaminated soil |
| CN109402188A (en) * | 2018-10-19 | 2019-03-01 | 江南大学 | A kind of ω-transaminase from bacillus pumilus and the application in biological amination |
| CN111951898A (en) * | 2020-06-30 | 2020-11-17 | 中国科学院微生物研究所 | Method for screening 2-ketolase capable of converting L-amino acid into |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733958A (en) * | 2016-03-16 | 2016-07-06 | 湖南省环境保护科学研究院 | Fusarium oxysporum and application thereof to phytoremediation of heavy metal contaminated soil |
| CN105733958B (en) * | 2016-03-16 | 2018-03-13 | 湖南省环境保护科学研究院 | A kind of Fusarium oxysporum and its application in Phytoremediation of Soils Contaminated by Heavy Metals |
| CN109402188A (en) * | 2018-10-19 | 2019-03-01 | 江南大学 | A kind of ω-transaminase from bacillus pumilus and the application in biological amination |
| CN111951898A (en) * | 2020-06-30 | 2020-11-17 | 中国科学院微生物研究所 | Method for screening 2-ketolase capable of converting L-amino acid into |
| CN111951898B (en) * | 2020-06-30 | 2024-02-23 | 中国科学院微生物研究所 | Method for screening enzyme capable of converting L-amino acid into 2-keto acid |
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