CN106754806A - A kind of improved transaminase and its application in the preparation of (R) 3 amino butanol - Google Patents
A kind of improved transaminase and its application in the preparation of (R) 3 amino butanol Download PDFInfo
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Abstract
The present invention relates to a kind of improved transaminase, encoding gene and its application in the preparation of (R) 3 amino butanol.Enzymatic activity of the enzymatic activity of the improved transaminase higher than wild type transaminase, the amino acid sequence of wild type transaminase is as shown in SEQ ID No.2, from aspergillus terreus Aspergillus terreus NIH2624, improved transaminase includes the amino acid sequence for having at least 95% sequence homology with amino acid sequence shown in SEQ ID No.2, and is proline residue corresponding to the 215th in amino acid sequence shown in SEQ ID No.2.The reaction condition that wherein improved transaminase is used to prepare (R) 3 amino butanol is gentle, and product chiral purity and yield are higher, and concentration of substrate is up to 50g/L, with preferable prospects for commercial application.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to a kind of improved transaminase, encoding gene and its in (R) -3-
Application prepared by amino butanol.
Background technology
(R) -3- amino butanols have a wide range of applications in terms of chemical reaction and pharmaceutical synthesis, can be used as various chiral medicines
The key intermediate of thing.Such as inverase Du Lutewei (Dolutegravir), the medicine is researched and developed by GlaxoSmithKline PLC (GSK)
Production, the food and drug administrations of in 8Yue12Huo, 2013 (FDA) approval listing.Du Lutewei has been sold since the listing
Volume is presented rapid development, and 2015 annual global sales volumes are up to 9,000,000,000 dollars.
(R) under -3- amino butanols normal temperature and pressure it is a kind of colourless thick liquid, molecular weight is 89.14, water-soluble, second
In alcohol, ethyl acetate equal solvent, its structural formula is as follows：
Based on chemical method, one kind is to use Kinetic Resolution to the synthetic method of (the R) -3- amino butanols announced at present
Obtain the 3- amino butanols (Journal of Organic Chemistry, 42,1650,1977) of chiral purity, the method product
Yield is low, and course of reaction is using Lithium Aluminium Hydride as reducing agent, and industrial amplification production is relatively hazardous.Another kind is with chiral compound
Thing obtains the 3- amino butanols of chiral purity by multistep reaction as initiation material, such as D-alanine or R types phenyl ethylamine
(Tetrahedron, 61,9031,2005 and CN 101417954B), chiral purity cost of material is more expensive in this class method, and instead
Answer step tediously long, production cost is higher.
Compared with chemical synthesis, biological synthesis process has reaction condition gentle, high conversion rate and stereoselectivity strong etc.
Advantage, the report for preparing (R) -3- amino butanols currently with bioanalysis is also less.Transaminase being capable of the latent chirality of asymmetry catalysis
Ketone compounds one-step synthesis chirality aminated compounds, with preferable application prospect.Patent CN 104131048A disclose one
Plant the method for synthesizing (R) -3- amino butanols using the butanone of wild type D- transaminase asymmetry catalysis 4- hydroxyls -2, but its reaction bottom
Thing concentration is only up to 300mM (26.4g/L), it is impossible to meet industrial production demand, only rests on the laboratory research stage, together
When course of reaction in need use 10% organic cosolvent DMSO or acetonitrile, which increases post-reaction treatment operation and into
This, three industrial wastes discharge capacity is increased.
The content of the invention
In order to overcome the drawbacks described above existing for prior art, the invention provides a kind of improved transaminase, while also
Disclose encoding gene, recombination expression genetic engineering bacterium and its application in the preparation of (R) -3- amino butanols of the transaminase.
First aspect present invention provides a kind of improved transaminase, enzyme activity of its enzymatic activity higher than wild type transaminase
Property.
The enzymatic activity refers to that catalyzing ketone compound occurs the enzymatic activity that asymmetric transamination reaction generates Chiral Amine.
The wild type transaminase amino acid sequence as shown in SEQ ID No.2, and from aspergillus terreus
Aspergillus terreus NIH2624。
In one particular embodiment of the present invention, the enzymatic activity refers to that catalytic substrate 4- hydroxy-2-butanones occur not
Symmetrical transamination reaction generates the enzymatic activity of (R) -3- amino butanols.
The improved transaminase is by substitution, missing or addition one in amino acid sequence shown in SEQ ID No.2
The derivative protein with transaminase-catalyzed activity of individual or several amino acid residues, and with amino acid shown in SEQ ID No.2
Sequence has at least 95% sequence homology.Wherein, described " several " refer to 2-15, more preferably less than 10.According to this hair
It is bright, being mutated for 2,5,10 amino acid residues is carried out in the protein molecule of amino acid sequence as shown in SEQ ID No.2
Protein to amino acid sequence shown in SEQ ID No.4,6,8 is respectively provided with the activity of transaminase higher.
The SEQ ID No.4 are the ammonia for having at least 95% sequence homology with amino acid sequence shown in SEQ ID No.2
Base acid sequence, and it is respectively alanine and dried meat corresponding to the 55th in amino acid sequence shown in SEQ ID No.2 and the 215th
Propylhomoserin.
The SEQ ID No.6 are the ammonia for having at least 95% sequence homology with amino acid sequence shown in SEQ ID No.2
Base acid sequence, and corresponding to the 54th in amino acid sequence shown in SEQ ID No.2, the 55th, the 117th, the 161st and
215th is respectively tyrosine, serine, alanine, leucine and proline.
The SEQ ID No.8 are the ammonia for having at least 95% sequence homology with amino acid sequence shown in SEQ ID No.2
Base acid sequence, and corresponding to the 54th in amino acid sequence shown in SEQ ID No.2, the 55th, the 58th, the 117th,
126, the 142nd, the 144th, the 161st, the 207th and the 215th are respectively tyrosine, serine, alanine, the third ammonia
Acid, proline, serine, cysteine, leucine, cysteine and proline.
In one particular embodiment of the present invention, the amino acid sequence of described improved transaminase is SEQ ID
No.4。
In one particular embodiment of the present invention, the enzymatic activity of the improved transaminase is the wild type transaminase enzyme
At least 2 times of activity.
Second aspect present invention provides the gene of the above-mentioned improvement transaminase of coding, its include with shown in SEQ ID No.1
Nucleotides sequence show the nucleotide sequence of at least 95% sequence homology.
The nucleotide sequence of the encoding gene of the improved transaminase may be selected from SEQ ID No.3, SEQ ID No.5 and
SEQ ID No.7.
In one particular embodiment of the present invention, the nucleotide sequence of the encoding gene of the improved transaminase such as SEQ
Shown in ID No.3.
Third aspect present invention provides a kind of genetic engineering bacterium, and the strain expresses above-mentioned improved transaminase.
In one particular embodiment of the present invention, the expression vector that the genetic engineering bacterium is used is pET-24a, host
Cell is e. coli bl21 (DE3).
It is not right in catalyzing ketone compound that fourth aspect present invention provides a kind of improved transaminase of the present invention
Claim the application in transamination reaction generation chiral amine compound.
In above-mentioned application, each condition of the reaction can be selected by the normal condition of the such reaction in this area, preferably
It is as follows：
In the buffer solution of pH=7-9, add respectively a certain proportion of isopropylamine, phosphopyridoxal pyridoxal phosphate (PLP), 4- hydroxyls-
2- butanone and improved transaminase of the present invention, carry out asymmetric transamination reaction at 30-40 DEG C, generate optically active
(R) -3- amino butanols.In reaction mixture the concentration of transaminase be 5-10g/L, isopropylamine concentration be 60-90g/L, 4- hydroxyl
The concentration of base -2- butanone is the concentration of 25-50g/L, PLP for the concentration of 0.1-1mM and buffer solution is 10-100mM.During reaction
Between in course of reaction the production concentration no longer increased time be defined.After reaction terminates, can be by this area conventional method from reaction
Product (R) -3- amino butanols are extracted in liquid.
The improved transaminase is obtained by engineering bacteria fermentation, with enzyme powder, the clasmatosis liquid of transaminase or is contained
The form of the somatic cells of ketoreductase is added, and is preferably added in enzyme powder form.
In one particular embodiment of the present invention, transaminase enzyme powder can use the conventional molecular biology in this area, surpass
Sound is broken and vacuum freeze-drying method is obtained.
Compared with prior art, the invention provides a kind of catalysis activity it is high, enantioselectivity is strong, substrate tolerance good
Improved transaminase, and using the method for transaminase-catalyzed synthesis (the R) -3- amino butanols.The method reaction condition is gentle,
Product chiral purity and yield are higher, while concentration of substrate is up to 50g/L, substantially increase the preparation of (R) -3- amino butanols
Efficiency, reduces its production cost, with preferable prospects for commercial application.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of mutator fusion DNA vaccine product in the embodiment of the present invention 1；
Fig. 2 is the polyacrylamide of expression AtATmut transaminase clasmatosis precipitations and supernatant in the embodiment of the present invention 3
Gel electrophoresis figure.A is broken precipitation, and B is broken supernatant.
Specific embodiment
Technology contents of the invention are further elaborated with reference to specific embodiment, its purpose is to more preferable
Understand present disclosure, but protection scope of the present invention not limited to this.
The structure of the transaminase mutant library of embodiment 1
Full genome composition sequence nucleotides as shown in SEQ ID No.1, two restriction enzyme sites of selection NdeI and HindIII are inserted
Enter pET24a expression vectors, the recombinant expression carrier of acquisition is named as pET24a-AtAT.To build mutant library, we design
Following 6 primers, refer to table 1：
The PCR primer table of table 1
Enter performing PCR using above-mentioned primer as template with pET24a-AtAT to expand, PCR system is：10 × PCR buffer are
5uL, 2.5mM dNTP are 4uL, and pfu DNA Polymerse are 0.5uL, and pET24a-AtAT templates are that 0.5uL (contains DNA profiling
0.2ug), ddH2O is 36uL, respectively with AtAT-up sense primers in table 1 (SEQ ID No.9) and H55-down anti-sense primers
(SEQ ID No.12), H55-up sense primers (SEQ ID No.11) and S215-down anti-sense primers (SEQ ID No.14),
S215-up sense primers (SEQ ID No.13) and AtAT-down anti-sense primers (SEQ ID No.10) each 2uL (10umol/L)
Enter performing PCR amplification.PCR amplification steps are：(1) 95 DEG C, predegeneration 3min；(2) 95 DEG C, it is denatured 30s；(3) 55 DEG C of annealing 30s；
(4) 72 DEG C of extension 1min；Step (2)~(4) repeat 30 times；(5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.PCR primer is passed through
Agarose gel electrophoresis reclaims 160 after purification, 480, the target stripe of 320bp or so.With these PCR primers as template, reaction
System is as follows：10 × PCR buffer are 5uL, and 2.5mM dNTP are 4uL, and pfu DNA Polymerse are 0.5uL, and ddH2O is
33.5uL, PCR reclaim each 1uL of fragment, enter performing PCR using AtAT-up sense primers and each 2uL of AtAT-down anti-sense primers and expand
Increase, PCR amplification steps are：(1) 95 DEG C, predegeneration 3min；(2) 95 DEG C, it is denatured 30s；(3) 55 DEG C of annealing 30s；(4) 72 DEG C are prolonged
Stretch 70s；Step (2)~(4) repeat 30 times；(5) 72 DEG C are continued to extend 10min, are cooled to 4 DEG C.PCR primer is through Ago-Gel
The target stripe (see Fig. 1) of 1000bp or so is reclaimed after Purified in electrophoresis, is then contained in the fragment prominent with two Sites Combinations
The all mutant nucleotide sequences for becoming.By said gene fragment and carrier pET-24a plasmids carry out respectively endonuclease reaction (NdeI and
HindIII, 37 DEG C of digestion 1h), coupled reaction (16 DEG C of reaction overnights) is carried out after digestion products gel extraction, it is transferred to E.coli
BL21 (DE3) competent cell, is screened by kanamycins and obtains positive monoclonal.Added per hole in 96 orifice plates LB+ cards that
Mycin culture medium 0.6mL, every piece of 96 orifice plates select 93 positive colonies and 3 BL21 (DE3)/pET24a-AtAT as control,
6 piece of 96 orifice plate of common picking, the concussion and cultivate 16h in 37 DEG C of shaking tables, this is mutant library.
The expression and screening of the transaminase mutant of embodiment 2
The mutant library of overnight incubation is taken into 100uL bacterium solutions per hole to be inoculated with into a 96 new orifice plates, the plate is included per hole
Fresh LB+ kanamycins culture medium 1mL, shaking table culture is to OD under the conditions of 37 DEG C600Value reaches 0.8~1.0, is subsequently added
IPTG to final concentration of 1.0mM, in 25 DEG C of Fiber differentiations 20 hours or so, supernatant, collects thalline are abandoned in centrifugation.Added per hole
400uL reaction solutions, including：The substrate 4- hydroxy-2-butanones of 20g/L, the isopropylamine of 60g/L, the coenzyme PLP of 1mM,
100mM kaliumphosphate buffers (pH=8.0), pH is adjusted in 10M NaOH after 30 DEG C of shaking table oscillating reactions 24h, are added per hole>13,
Isometric ethyl acetate extraction is added, after centrifugation, drawing organic layer carries out GC analyses, detects mutant enzyme activity.GC conditions：
Agilent 7820A gas chromatographs；RESTEK(30m*0.25mm*0.25 μm) capillary column；Hydrogen flame
Ionization detector；220 DEG C of detector temperature；200 DEG C of injector temperature；Temperature programming, initial temperature be 100 DEG C, 10 DEG C/
Min rises to 200 DEG C, keeps 15min；Input mode is split sampling, split ratio 50:1.Detected by GC and obtain a plant mutant body
Conversion ratio>95%, and control group conversion ratio<20%.In order to obtain transaminase from the cell of expression mutant is filtered out
Encoding gene, sequencing is sent by the strain corresponding to it, and sequencing result shows containing nucleotide sequence shown in SEQ ID No.3, compiles
Amino acid sequence shown in code SEQID No.4.The improved transaminase is named as AtATmut, strain be named as BL21 (DE3)/
pET24a-AtATmut。
It is prepared by the expression of the AtATmut transaminases of embodiment 3 and enzyme powder
LB Tube propagation bases of switching glycerol stocks strain BL21 (the DE3)/pET24a-AtATmut to 5mL containing kanamycins
Middle activation culture (37 DEG C of culture 12h), is trained by 1% inoculum concentration switching activation culture thing to LB liquid of the 400mL containing kanamycins
Support in base, 37 DEG C of culture OD to 0.6-0.8 add IPTG (final concentration 0.1mM) in 25 DEG C of Fiber differentiation 16h.Bacterium is collected by centrifugation
Body, with 40mL phosphate buffers (10mM, pH 7.5) resuspended thalline after, the ultrasonication 15min in ice-water bath is collected by centrifugation
Supernatant is precipitated, and SDS-PAGE detection display AtATmut transaminases exist (see Fig. 2) in partly soluble form, supernatant -20
Pulverized after vacuum freeze drying 48h after DEG C pre-freeze, obtain final product AtATmut transaminase enzyme powders.
The asymmetric transamination reaction of the transaminase-catalyzed 4- hydroxy-2-butanones of the AtATmut of embodiment 4
The phosphate buffer (40mL, pH=7.5) and isopropylamine (4.5g) of 100mM are added in 100mL reaction vessels,
With phosphorus acid for adjusting pH to 7.5, substrate 4- hydroxy-2-butanones (2.5g) is added, be stirring evenly and then adding into AtATmut enzyme powders
(0.25g) and coenzyme PLP (13.25mg), is settled to 50mL, and the open reaction of magnetic agitation, uses isopropyl at 35 DEG C in course of reaction
Amine (4M) control reaction pH detects reaction process in 7.5 or so, TLC.80 DEG C of insulation 2h make albumen in reaction solution after reaction terminates
Qualitative change, filtering goes isolating protein, plus NaOH (10M) regulation pH=13, isometric ethyl acetate to extract three times, merge organic
Phase, anhydrous sodium sulfate drying, vacuum distillation obtains (R) -3- amino butanols (2.3g), product yield 91%.GC detects conversion ratio
It is that 99%, R type product ee values are 99.5%.
The asymmetric transamination reaction of the transaminase-catalyzed 4- hydroxy-2-butanones of the AtATmut of embodiment 5
The phosphate buffer (40mL, pH=9.0) and isopropylamine (3.0g) of 10mM are added in 100mL reaction vessels,
With phosphorus acid for adjusting pH to 8.0, substrate 4- hydroxy-2-butanones (2.0g) is added, be stirring evenly and then adding into AtATmut enzyme powders (0.5g)
With coenzyme PLP (13.25mg), 50mL is settled to, the open reaction of magnetic agitation, is controlled in course of reaction with isopropylamine (4M) at 40 DEG C
System reaction pH detects reaction process in 8.0 or so, TLC.80 DEG C of insulation 2h make protein denaturation in reaction solution, mistake after reaction terminates
Isolating protein, plus NaOH (10M) regulation pH=13 are filtered off, isometric ethyl acetate is extracted three times, merges organic phase, anhydrous slufuric acid
Sodium is dried, and vacuum distillation obtains (R) -3- amino butanols (1.93g), product yield 90%.GC detection conversion ratios are 99%, R types
Product ee values are 99.5%.
The asymmetric transamination reaction of the transaminase-catalyzed 4- hydroxy-2-butanones of the AtATmut of embodiment 6
The phosphate buffer (40mL, pH=7.0) and isopropylamine (3.0g) of 10mM are added in 100mL reaction vessels,
With phosphorus acid for adjusting pH to 7.0, substrate 4- hydroxy-2-butanones (1.25g) is added, be stirring evenly and then adding into AtATmut enzyme powders
(0.5g) and coenzyme PLP (1.325mg), is settled to 50mL, and the open reaction of magnetic agitation, uses isopropylamine at 30 DEG C in course of reaction
(4M) control reaction pH detects reaction process in 7.0 or so, TLC.80 DEG C of insulation 2h make protein in reaction solution after reaction terminates
Denaturation, filtering goes isolating protein, plus NaOH (10M) regulation pH=13, isometric ethyl acetate to extract three times, merges organic phase,
Anhydrous sodium sulfate drying, vacuum distillation obtains (R) -3- amino butanols (1.15g), product yield 92%.GC detects that conversion ratio is
99%, R type product ee values are 99.5%.
SEQUENCE LISTING
<110>Still section's biological medicine（Shanghai）Co., Ltd
<120>A kind of improved transaminase and its application in the preparation of (R) -3- amino butanols
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 978
<212> DNA
<213>Artificial sequence
<400> 1
atggcttcta tggacaaagt tttcgctggt tacgctgctc gtcaggctat cctggaatct 60
accgaaacca ccaacccgtt cgctaaaggt atcgcttggg ttgaaggtga actggttccg 120
ctggctgaag ctcgtatccc gctgctggac cagggtttca tgcactctga cctgacctac 180
gacgttccct ctgtgtggga cgggaggttc ttccgtctgg acgaccacat cacccgtctg 240
gaagctagtt gcaccaaact gcgtctgcgt ctgccgctgc cgcgtgacca ggttaaacag 300
atcctggttg aaatggttgc taaatctggt atccgtgacg ctttcgttga actgatcgtt 360
acccgtggtc tgaaaggtgt tcgtggtacc cgtccggaag acatcgttaa caacctgtac 420
atgttcgttc agccatacgt gtgggtcatg gaaccggaca tgcagcgtgt tggtggttct 480
gctgttgttg ctcgtaccgt tcgtcgtgtt ccgccgggtg ctatcgaccc gaccgttaaa 540
aacctgcagt ggggtgacct ggttcgtggt atgttcgaag ctgctgaccg tggtgctacc 600
tacccgttcc tgaccgacgg tgacgctcac ctgaccgaag gttctggttt caacatcgtt 660
ctggttaaag acggtgttct gtacaccccg gaccgtggtg ttctgcaggg tgttacccgt 720
aaatctgtta tcaacgctgc tgaagcgttc ggtatcgaag ttcgtgttga attcgttccg 780
gttgaactgg cttaccgttg cgacgaaatc ttcatgtgca ccaccgctgg tggtatcatg 840
ccgatcacca ccctggacgg tatgccggtt aacggtggtc agatcggtcc gatcaccaaa 900
aaaatctggg acggttactg ggctatgcac tacgacgctg cttactcttt cgaaatcgac 960
tacaacgaac gtaactaa 978
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Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
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Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
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Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
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Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
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Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
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Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
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Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
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atggcttcta tggacaaagt tttcgctggt tacgctgctc gtcaggctat cctggaatct 60
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ctggctgaag ctcgtatccc gctgctggac cagggtttca tggcgtctga cctgacctac 180
gacgttccct ctgtgtggga cgggaggttc ttccgtctgg acgaccacat cacccgtctg 240
gaagctagtt gcaccaaact gcgtctgcgt ctgccgctgc cgcgtgacca ggttaaacag 300
atcctggttg aaatggttgc taaatctggt atccgtgacg ctttcgttga actgatcgtt 360
acccgtggtc tgaaaggtgt tcgtggtacc cgtccggaag acatcgttaa caacctgtac 420
atgttcgttc agccatacgt gtgggtcatg gaaccggaca tgcagcgtgt tggtggttct 480
gctgttgttg ctcgtaccgt tcgtcgtgtt ccgccgggtg ctatcgaccc gaccgttaaa 540
aacctgcagt ggggtgacct ggttcgtggt atgttcgaag ctgctgaccg tggtgctacc 600
tacccgttcc tgaccgacgg tgacgctcac ctgaccgaag gtcctggttt caacatcgtt 660
ctggttaaag acggtgttct gtacaccccg gaccgtggtg ttctgcaggg tgttacccgt 720
aaatctgtta tcaacgctgc tgaagcgttc ggtatcgaag ttcgtgttga attcgttccg 780
gttgaactgg cttaccgttg cgacgaaatc ttcatgtgca ccaccgctgg tggtatcatg 840
ccgatcacca ccctggacgg tatgccggtt aacggtggtc agatcggtcc gatcaccaaa 900
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<210> 4
<211> 325
<212> PRT
<213>Artificial sequence
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Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Met Ala Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Pro Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
<210> 5
<211> 978
<212> DNA
<213>Artificial sequence
<400> 5
atggcttcta tggacaaagt tttcgctggt tacgctgctc gtcaggctat cctggaatct 60
accgaaacca ccaacccgtt cgctaaaggt atcgcttggg ttgaaggtga actggttccg 120
ctggctgaag ctcgtatccc gctgctggac cagggtttct actcttctga cctgacctac 180
gacgttccct ctgtgtggga cgggaggttc ttccgtctgg acgaccacat cacccgtctg 240
gaagctagtt gcaccaaact gcgtctgcgt ctgccgctgc cgcgtgacca ggttaaacag 300
atcctggttg aaatggttgc taaatctggt atccgtgacg ctttcgttgc tctgatcgtt 360
acccgtggtc tgaaaggtgt tcgtggtacc cgtccggaag acatcgttaa caacctgtac 420
atgttcgttc agccatacgt gtgggtcatg gaaccggaca tgcagcgtgt tggtggttct 480
ctggttgttg ctcgtaccgt tcgtcgtgtt ccgccgggtg ctatcgaccc gaccgttaaa 540
aacctgcagt ggggtgacct ggttcgtggt atgttcgaag ctgctgaccg tggtgctacc 600
tacccgttcc tgaccgacgg tgacgctcac ctgaccgaag gtccgggttt caacatcgtt 660
ctggttaaag acggtgttct gtacaccccg gaccgtggtg ttctgcaggg tgttacccgt 720
aaatctgtta tcaacgctgc tgaagcgttc ggtatcgaag ttcgtgttga attcgttccg 780
gttgaactgg cttaccgttg cgacgaaatc ttcatgtgca ccaccgctgg tggtatcatg 840
ccgatcacca ccctggacgg tatgccggtt aacggtggtc agatcggtcc gatcaccaaa 900
aaaatctggg acggttactg ggctatgcac tacgacgctg cttactcttt cgaaatcgac 960
tacaacgaac gtaactaa 978
<210> 6
<211> 325
<212> PRT
<213>Artificial sequence
<400> 6
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Tyr Ser Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Ala Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Leu Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Pro Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
<210> 7
<211> 978
<212> DNA
<213>Artificial sequence
<400> 7
atggcttcta tggacaaagt tttcgctggt tacgctgctc gtcaggctat cctggaatct 60
accgaaacca ccaacccgtt cgctaaaggt atcgcttggg ttgaaggtga actggttccg 120
ctggctgaag ctcgtatccc gctgctggac cagggtttct actcttctga cgctacctac 180
gacgttccct ctgtgtggga cgggaggttc ttccgtctgg acgaccacat cacccgtctg 240
gaagctagtt gcaccaaact gcgtctgcgt ctgccgctgc cgcgtgacca ggttaaacag 300
atcctggttg aaatggttgc taaatctggt atccgtgacg ctttcgttgc tctgatcgtt 360
acccgtggtc tgaaaccggt tcgtggtacc cgtccggaag acatcgttaa caacctgtac 420
atgtctgttt gcccatacgt gtgggtcatg gaaccggaca tgcagcgtgt tggtggttct 480
ctggttgttg ctcgtaccgt tcgtcgtgtt ccgccgggtg ctatcgaccc gaccgttaaa 540
aacctgcagt ggggtgacct ggttcgtggt atgttcgaag ctgctgaccg tggtgctacc 600
tacccgttcc tgaccgactg cgacgctcac ctgaccgaag gtccgggttt caacatcgtt 660
ctggttaaag acggtgttct gtacaccccg gaccgtggtg ttctgcaggg tgttacccgt 720
aaatctgtta tcaacgctgc tgaagcgttc ggtatcgaag ttcgtgttga attcgttccg 780
gttgaactgg cttaccgttg cgacgaaatc ttcatgtgca ccaccgctgg tggtatcatg 840
ccgatcacca ccctggacgg tatgccggtt aacggtggtc agatcggtcc gatcaccaaa 900
aaaatctggg acggttactg ggctatgcac tacgacgctg cttactcttt cgaaatcgac 960
tacaacgaac gtaactaa 978
<210> 8
<211> 325
<212> PRT
<213>Artificial sequence
<400> 8
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Tyr Ser Ser Asp Ala Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Ala Leu Ile Val Thr Arg Gly Leu Lys Pro Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Ser Val Cys
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Leu Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Cys Asp
195 200 205
Ala His Leu Thr Glu Gly Pro Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
<210> 9
<211> 31
<212> DNA
<213>Artificial sequence
<400> 9
cccaagcttt tagttacgtt cgttgtagtc g 31
<210> 10
<211> 30
<212> DNA
<213>Artificial sequence
<400> 10
gggaattcca tatggcttct atggacaaag 30
<210> 11
<211> 32
<212> DNA
<213>Artificial sequence
<400> 11
ctggaccagg gtttcatgvn ctctgacctg ac 32
<210> 12
<211> 29
<212> DNA
<213>Artificial sequence
<400> 12
cgtaggtcag gtcagagnbc atgaaaccc 29
<210> 13
<211> 30
<212> DNA
<213>Artificial sequence
<400> 13
tcacctgacc gaaggtnnkg gtttcaacat 30
<210> 14
<211> 29
<212> DNA
<213>Artificial sequence
<400> 14
gaacgatgtt gaaaccmnna ccttcggtc 29
Claims (8)
1. a kind of improved transaminase, it is characterised in that：
Enzymatic activity of the enzymatic activity of the improved transaminase higher than wild type transaminase；
The enzymatic activity refers to that catalyzing ketone compound occurs the enzymatic activity that asymmetric transamination reaction generates Chiral Amine；
The amino acid sequence of the wild type transaminase as shown in SEQ ID No.2, and from aspergillus terreus Aspergillus
terreus NIH2624；
The improved transaminase includes the amino for having at least 95% sequence homology with amino acid sequence shown in SEQ ID No.2
Acid sequence, and be proline residue corresponding to the 215th in amino acid sequence shown in SEQ ID No.2.
2. as claimed in claim 1 improved transaminase, it is characterised in that：The enzymatic activity refers to catalysis 4- hydroxy-2-butanones
There is the enzymatic activity that asymmetric transamination reaction generates (R) -3- amino butanols.
3. as claimed in claim 1 improved transaminase, it is characterised in that：The amino acid sequence of the improved transaminase is selected from
SEQ ID No.4, SEQ ID No.6 or SEQ ID No.8.
4. as claimed in claim 1 improved transaminase, it is characterised in that：The enzymatic activity of the improved transaminase is the open country
At least 2 times of raw type transaminase enzymatic activity.
5. a kind of gene for encoding improved transaminase as described in claim any one of 1-4, it is characterised in that：The gene
Nucleotide sequence be selected from SEQ ID No.3, SEQ ID No.5 or SEQ ID No.7.
6. a kind of genetic engineering bacterium, it is characterised in that：Improved turn in strain expression claim 1-4 described in any one
Ammonia enzyme.
7. genetic engineering bacterium as claimed in claim 6, it is characterised in that：The expression vector that the genetic engineering bacterium is used is
PET-24a, host cell is e. coli bl21 (DE3).
8. the improved transaminase as described in claim any one of 1-4 occurs that asymmetric to turn ammonia anti-in catalysis 4- hydroxy-2-butanones
The application in (R) -3- amino butanols should be generated.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611181535.9A CN106754806B (en) | 2016-12-20 | 2016-12-20 | Improved transaminase and application thereof in preparation of (R) -3-aminobutanol |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611181535.9A CN106754806B (en) | 2016-12-20 | 2016-12-20 | Improved transaminase and application thereof in preparation of (R) -3-aminobutanol |
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| Publication Number | Publication Date |
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| CN106754806A true CN106754806A (en) | 2017-05-31 |
| CN106754806B CN106754806B (en) | 2020-04-14 |
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|---|---|---|---|
| CN201611181535.9A Active CN106754806B (en) | 2016-12-20 | 2016-12-20 | Improved transaminase and application thereof in preparation of (R) -3-aminobutanol |
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| CN107653233A (en) * | 2017-07-06 | 2018-02-02 | 泰州学院 | A kind of improved transaminase, its encoding gene and the genetic engineering bacterium for expressing the enzyme |
| CN108823179A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of transaminase from actinomyces, mutant, recombinant bacterium and application |
| CN109486787A (en) * | 2018-12-23 | 2019-03-19 | 尚科生物医药（上海）有限公司 | A kind of stability-enhanced transaminase mutant of pH |
| CN109957554A (en) * | 2017-12-26 | 2019-07-02 | 宁波酶赛生物工程有限公司 | It is engineered TRANSAMINASE POLYPEPTIDES and its application |
| CN110358804A (en) * | 2018-04-10 | 2019-10-22 | 湖州颐辉生物科技有限公司 | The enzymatic production process of R-3- amino n-butanol |
| CN111363732A (en) * | 2020-03-12 | 2020-07-03 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus NIH2624 and application thereof |
| CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
| CN112280761A (en) * | 2020-11-16 | 2021-01-29 | 清华大学 | Recombinant transaminase, mutant of recombinant transaminase and application of mutant |
| CN114075557A (en) * | 2020-08-19 | 2022-02-22 | 上海飞腾医药科技有限公司 | Recombinant transaminase and application thereof in synthesis of (R) -2- (2, 5-difluorophenyl) pyrrolidine |
| CN116515788A (en) * | 2023-03-02 | 2023-08-01 | 斯芬克司药物研发（天津）股份有限公司 | Novel R-type omega-aminotransferase and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653233A (en) * | 2017-07-06 | 2018-02-02 | 泰州学院 | A kind of improved transaminase, its encoding gene and the genetic engineering bacterium for expressing the enzyme |
| CN109957554A (en) * | 2017-12-26 | 2019-07-02 | 宁波酶赛生物工程有限公司 | It is engineered TRANSAMINASE POLYPEPTIDES and its application |
| CN110358804A (en) * | 2018-04-10 | 2019-10-22 | 湖州颐辉生物科技有限公司 | The enzymatic production process of R-3- amino n-butanol |
| CN108823179B (en) * | 2018-06-30 | 2020-10-09 | 浙江工业大学 | Transaminase derived from actinomycetes, mutant, recombinant bacterium and application |
| CN108823179A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of transaminase from actinomyces, mutant, recombinant bacterium and application |
| CN109486787A (en) * | 2018-12-23 | 2019-03-19 | 尚科生物医药（上海）有限公司 | A kind of stability-enhanced transaminase mutant of pH |
| CN111363732B (en) * | 2020-03-12 | 2023-05-23 | 卡柔恩赛生物技术湖北有限公司 | Transaminase mutant from aspergillus terreus NIH2624 and application thereof |
| CN111363732A (en) * | 2020-03-12 | 2020-07-03 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus NIH2624 and application thereof |
| CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
| CN111549011B (en) * | 2020-06-03 | 2023-05-23 | 卡柔恩赛生物技术湖北有限公司 | Transaminase mutant from aspergillus terreus and application thereof |
| CN114075557A (en) * | 2020-08-19 | 2022-02-22 | 上海飞腾医药科技有限公司 | Recombinant transaminase and application thereof in synthesis of (R) -2- (2, 5-difluorophenyl) pyrrolidine |
| CN114075557B (en) * | 2020-08-19 | 2023-12-22 | 上海飞腾医药科技有限公司 | Recombinant aminotransferase and its use in the synthesis of (R) -2- (2, 5-difluorophenyl) pyrrolidine |
| CN112280761A (en) * | 2020-11-16 | 2021-01-29 | 清华大学 | Recombinant transaminase, mutant of recombinant transaminase and application of mutant |
| CN112280761B (en) * | 2020-11-16 | 2022-04-12 | 清华大学 | Recombinant transaminase, mutant of recombinant transaminase and application of mutant |
| CN116515788A (en) * | 2023-03-02 | 2023-08-01 | 斯芬克司药物研发（天津）股份有限公司 | Novel R-type omega-aminotransferase and application thereof |
| CN116515788B (en) * | 2023-03-02 | 2023-09-12 | 斯芬克司药物研发（天津）股份有限公司 | R-type omega-aminotransferase and application thereof |
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