CN110016467A - Carbonyl reductase ChKRED20 mutant and application thereof - Google Patents
Carbonyl reductase ChKRED20 mutant and application thereof Download PDFInfo
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Abstract
The invention belongs to genetic engineerings and technical field of enzyme engineering, and in particular to a kind of carbonyl reduction enzyme mutant and application thereof.Carbonyl reductase ChKRED20 can be with the chloro- 1- (3 of asymmetric reduction 2-, 4- difluorophenyl) ethyl ketone obtains the chloro- 1- (3 of ticagrelor intermediate (S) -2-, 4- difluorophenyl) ethyl alcohol (e.e. value > 99%), evolution is oriented to the enzyme molecule using fallibility round pcr and single-point saturation mutation technology, the mutant that 11 enzyme activity improve 1.6-10 times is obtained, it is shown that the application potential in biocatalysis.
Description
Technical field
The invention belongs to genetic engineerings and technical field of enzyme engineering, and in particular to a kind of carbonyl reduction enzyme mutant and its use
On the way.
Background technique
Optical activity (S) -2- chloro- 1- (3,4- difluorophenyl) ethyl alcohol is the drug ticagrelor of Astrazeneca AB's research and development
The crucial chiral intermediate of (Ticagrelor, trade name " Brilinta ").Ticagrelor is first to be recognized in 2011 by FDA
The reversible P2Y of card12Receptor anticaking agent treats acute coronary syndrome for preventing artery sclerosis.
Ketoreductase (EC1.1.1.X, X=1,2) be also referred to as carbonyl reductase (Carbonyl reductase, CR) or
Alcohol dehydrogenase (Alcohol dehydrogenase, ADH), they are reversibly catalyzed ketone or aldehyde is reduced to alcohol, and need it is auxiliary because
The participation of son.Microbial cell or microbe-derived carbonyl reductase can efficiently be catalyzed the reduction of prochiral ketones, be
Prepare one of the important method of chiral alcohol molecule.Native enzyme in industrial application it is generally existing can not adapt to industrial process conditions and
The problems such as low to the catalytic capability of non-natural substrates.Be transformed by protein engineering method to enzyme molecule is to improve zymology
The effective means of energy.Carbonyl reductase ChKRED20 can be replaced with the chloro- 1- of asymmetric reduction 2- (3,4- difluorophenyl) ethyl ketone
Ge Ruiluo intermediate (S) -2- chloro- 1- (3,4- difluorophenyl) ethyl alcohol (e.e. value > 99%), using molecular evolution technique, into one
Step improves the catalysis activity of the enzyme, to push its industrial applications in intermediate production.
Summary of the invention
The present invention is using fallibility round pcr and single-point saturation mutation technology to from Chryseobacterium sp CA49
The carbonyl reductase ChKRED20 of (Chryseobacterium sp.CA49) carries out molecular modification, to obtain activity raising
Carbonyl reductase ChKRED20 mutant.
In order to reach the goals above, random mutation is carried out to female parent gene with fallibility round pcr first, establishes mutation text
Library goes out the prominent of 2 enzyme activity raisings by high flux screening System For Screening with the chloro- 1- of 2- (3,4- difluorophenyl) ethyl ketone for substrate
It conjugates point (H145L and L205M), and saturation mutation library is established respectively to the two sites, filter out the mutation of activity raising
Body.Wherein, the enzyme activity of muton L205A improves 10 times than maternal.
The present invention is achieved in that
(1) construction and screening of libraries of random mutants
Carbonyl reductase ChKRED20 derives from Chryseobacterium sp CA49 (Wuzhong willow, Liu Yan etc., one plant of Chryseobacterium sp and its carbonyl
Base reductase is produced for Aprepitant chiral intermediate, Chinese patent, 103497911 B of CN), which is
750bp encodes 249 amino acid, in the accession number of NCBI: KC342020.
Firstly, constructing libraries of random mutants using fallibility round pcr, obtain more than 2 × 104The mutant text of a clone
Library will extract plasmid after mutation library clone collection, be transferred to E. coli expression strains BL21-DE3, selects monoclonal in 96 orifice plates
Middle expression albumen.Then thalline were collected by centrifugation, and lysozyme smudge cells are added, and are centrifuged, take part supernatant (crude enzyme liquid) with
The chloro- 1- of 2- (3,4- difluorophenyl) ethyl ketone is substrate, and reaction appropriate time measures enzyme activity.Carbonyl reductase ChKRED20 is maternal
As a control group, the activity screened is higher than the bacterial strain of control, serves the sequencing of Hai Yingjun Bioisystech Co., Ltd, is dashed forward
Variant DNA sequences information.Detailed protocol is shown in example 1.
Through above-mentioned random mutation library construction and screening, the mutant that 2 activity improve are obtained, respectively H145L,
L205M, its feature is as follows:
H145L: the 145th Histidine mutagenesis of the enzyme is leucine (DNA sequence dna becomes CTT from CAT).
L205M: the 205th leucine of the enzyme sports methionine (DNA sequence dna becomes ATG from CTG).
(2) saturation mutation is carried out to the two sites
By designing degenerate primer, using site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) to carbonyl
The 145th, 205 site reductase ChKRED20 carries out saturation mutation respectively, obtains the mutant library of~1000 clones, will dash forward
Plasmid is extracted after becoming library clone collection, E. coli expression strains BL21-DE3 is transferred to, respectively selects 100 monoclonals in 96 orifice plates
Middle expression albumen carries out high flux screening.Further screen the mutant improved than maternal carbonyl reductase ChKRED20 enzyme activity
H145A, L205T, L205Q, L205S and L205A.
Since in the 145th saturation mutation library, the vigor of muton H145A is higher, therefore is with muton H145A
Female parent carries out saturation mutation in the 205th site, and same above-mentioned steps screen combinatorial mutagenesis H145A/L205A, H145A/
L205T, H145A/L205Q and H145A/L205V.
Mutant that above-mentioned vigor improves its feature is as follows:
H145A: the 145th Histidine mutagenesis of the enzyme is alanine (DNA sequence dna becomes GCT from CAT).
L205T: the 205th leucine of the enzyme sports threonine (DNA sequence dna becomes ACG from CTG).
L205Q: the 205th leucine of the enzyme sports glutamine (DNA sequence dna becomes CAG from CTG).
L205S: the 205th leucine of the enzyme sports serine (DNA sequence dna becomes AGC from CTG).
L205A: the 205th leucine of the enzyme sports alanine (DNA sequence dna becomes GCC from CTG).
H145A/L205A: the 145th Histidine mutagenesis of the enzyme is alanine (DNA sequence dna becomes GCT from CAT), the
205 leucines sport alanine (DNA sequence dna becomes GCG from CTG).
H145A/L205T: the 145th Histidine mutagenesis of the enzyme is alanine (DNA sequence dna becomes GCT from CAT), the
205 leucine MUTATION Threonines (DNA sequence dna becomes ACG from CTG).
H145A/L205Q: the 145th Histidine mutagenesis of the enzyme is alanine (DNA sequence dna becomes GCT from CAT), the
205 leucines sport glutamine (DNA sequence dna becomes CAG from CTG).
H145A/L205V: the 145th Histidine mutagenesis of the enzyme is alanine (DNA sequence dna becomes GCT from CAT), the
205 leucines sport valine (DNA sequence dna becomes GTG from CTG).
These mutant H145L, L205M, H145A, L205T, L205Q, L205S, L205A, H145A/L205A,
The activity of H145A/L205T, H145A/L205QH and H145A/L205V have not compared with maternal carbonyl reductase ChKRED20
It is improved with degree.Muton L205A activity promotes amplitude maximum, and stability and product e.e value are uninfluenced, and enzyme activity is
178.3 μm of ol/mg/min are 10 times of wild type.
The muton improved the invention has the advantages that: above-mentioned all enzyme activity compared with female parent, the reaction speed of enzyme faster, energy
Shorten reaction time, the spatiotemporal efficiency of catalysis substrate is improved, wherein the chloro- 1- of muton L205A conversion of substrate 2- (3,4- bis-
Fluorophenyl) spatiotemporal efficiency of ethyl ketone is up to 33.3g/ (L.h), there is good prospects for commercial application.
In addition to this, maternal carbonyl reductase ChKRED20 can at most convert concentration be 150g/L the chloro- 1- of substrate 2- (3,
4- difluorophenyl) ethyl ketone, and muton L205A can convert the substrate of 200g/L completely in 20h, e.e value it is constant (>
99%), conversion capability is greatly improved in muton biocatalysis, can further decrease production cost.
Detailed description of the invention
Fig. 1 is female parent carbonyl reductase ChKRED20 compared with the Rate activity of the mutant filtered out
Fig. 2 is the optimal reactive temperature measurement chart of female parent carbonyl reductase ChKRED20 and mutant L205A, maternal carbonyl
The optimal reactive temperature measurement curve of reductase ChKRED20 is indicated with；The optimal reactive temperature of mutant L205A measures bent
Line is indicated with ■.
Fig. 3 is the chloro- 1- of 2- of female parent carbonyl reductase ChKRED20 and mutant L205A conversion of substrate under the conditions of 40 DEG C
(3,4- difluorophenyl) ethyl ketone time graph, the concentration of maternal carbonyl reductase ChKRED20 conversion of substrate is respectively 100g/l
(zero), 150g/l () and 200g/l (◇)；And the conversion concentration of mutant L205A is 100g/l (●), 150g/l (■)
With 200g/l (◆).
Specific implementation method
Below in conjunction with embodiment, the present invention will be further described, it should be pointed out that the present embodiment is only used for explaining
The present invention, rather than limitation of the scope of the invention.
1 fallibility PCR of embodiment (error-prone PCR) method constructs carbonyl reductase libraries of random mutants
It utilizesII Random Mutagenesis kit carries out carbonyl reductase ChKRED20 gene
Random mutation.
The primer are as follows: T7:5 '-TAATACGACTCACTATAGGG -3 '
T7ter:5 '-TGCTAGTTATTGCTCAGCGG -3 '
Reaction condition are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 30s, 30s and 72 DEG C of 55 DEG C of annealing extension 90s, totally 25
It recycles, genetic fragment is recycled with plastic recovery kit after electrophoresis.
Will recycling segment with EcoR I and Sal I it is double digested after, pET 28a (+) carrier of digestion identical as process
(kalamycin resistance gene) is attached reaction, reaction condition are as follows: the ratio mixing of carrier and segment 1:3 in molar ratio add
Enter the T4DNA ligase of 400 units, 16 DEG C of connections overnight.Electric shocking method is transferred to Escherichia coli DH10B, obtains more than 2 × 104
The mutant library of a clone.
The screening of 2 carbonyl reductase ChKRED20 mutant library of embodiment
Plasmid will be extracted after mutation library clone collection in embodiment 1, be transferred to E. coli expression strains BL21-DE3, is coated with
LB plate containing kanamycin cultivates 12h.Picking monoclonal in 96 orifice plates, contain 200 μ L TB culture mediums and (contain 50 by every hole
μ g/mL kanamycins, 0.5mM IPTG), 30 DEG C, 180rpm, shake culture 18h.Each monoclonal is replicated with 96 orifice plate reproducers
After LB solid medium tablets, 37 DEG C of culture 12h, 4 DEG C of refrigerators are saved.96 orifice plates after thallus inducing expression are existed
It is centrifuged 10min under 4000rpm, is discarded supernatant, cell is resuspended in 200 μ L are added in each hole lysis buffer, and (lysis buffer is matched
System: the kaliumphosphate buffer of 0.1M, pH 8.0,10mg/mL lysozyme, 1 μ g/mL DNase I, 10mMMgCl2).It will be added with molten
96 orifice plates of bacterium solution are centrifuged 10min at 4000rpm after 37 DEG C of placement 60min, take supernatant for biocatalytic reaction.With
The supernatant (each 50 μ L) in each hole of 96 orifice plates is gently sucked out in the volley of rifle fire, then 150 μ L reaction solution (1mM are added in each hole of 96 orifice plates
NADH, the kaliumphosphate buffer of 0.1M, pH 8.0 and 0.02 times of volume contain the chloro- 1- of 1mM 2- (3,4- difluorophenyl) second
The dimethyl sulphoxide solution of ketone), after 30 DEG C of reaction 1h, the absorbance of NADH is measured at 340nm.In 96 orifice plate high flux screenings
In, acquisition vigor is higher by 50% bacterial strain H145L, L205M than wild type ChKRED20, (is implemented by further pure enzyme secondary screening
Example 3), measure its vigor respectively and be 3.1 times and 1.65 times of wild type control.It picks them separately monoclonal and serves extra large English fine horse biology skill
The sequencing of art company.
The measurement of 3 female parent of embodiment and mutant Rate activity
The preparation of 3.1 pure enzyme solutions
The purifying of maternal carbonyl reductase ChKRED20 and mutant H145L, L205M use affinity chromatography (Bio-
Rad)。
Picking monoclonal is into LB (the 50 μ g/mL containing kanamycins) culture medium, and 37 DEG C are incubated overnight, with 1% inoculum concentration
It is forwarded in TB (the 50 μ g/mL containing kanamycins) culture medium, 37 DEG C of culture 3h, after 0.5mMIPTG induction is added, 30 DEG C are continued to train
It supports to 18h.4 DEG C, thalline were collected by centrifugation by 6000rpm.It is resuspended in BufferA (50mM sodium phosphate buffer, pH 8.0,300mM
NaCl, 10mM imidazoles), cell homogeneous crusher machine has used supernatant addition later with 13000rpm, 4 DEG C of centrifugation 20min
In the column material of Buffer A balance, 30min is slightly mixed, rinses foreign protein with the Buffer A containing 20mM imidazoles, then with containing
The buffer of the Buffer A of 250mM imidazoles elutes destination protein, and is dialysed with sodium phosphate buffer and remove imidazoles (0.1M, pH
8.0), last electroresis appraisal purity.The measurement of protein concentration is measured using nucleic acid-protein micro quantitative determination instrument.
The measurement of 3.2 albumen Rate activities
Reaction system: volume accounts for the water phase of reaction system 70% and volume accounts for organic phase composition of reaction system 30%.Water
It mutually include the pure enzyme solution of 0.02mg/mL, the NAD of 0.2g/L+, the kaliumphosphate buffer of 0.1M, pH 8.0.Organic phase includes
The chloro- 1- of 20mM substrate 2- (3,4- difluorophenyl) ethyl ketone and isopropanol.40 DEG C, 150rpm reaction 5min.After reaction, bodies are waited
Product ethyl acetate extraction, measures the production quantity of product.
After measured, the vigor of mutant H145L, L205M is 3.1 times and 1.65 times of wild type control respectively.
Embodiment 4 constructs carbonyl reductase ChKRED20 saturation mutation library
Saturation mutation is carried out to the 145th, 205 site carbonyl reductase ChKRED20 respectively.Utilize NNS degenerate primer (N
Represent A, T, C, G；S represents G, C.), saturation library is respectively designated as SM145, SM205.
Meanwhile with muton H145A to be maternal, saturation mutation is carried out to 205 amino acids residues of the gene, is established
The library SM205/H145A.Used 145 and 205 degenerate primers it is as follows:
145S-F:5′-GAATATGGCCTCTATTNNSGGTATTGTTGCTGCACCGC-3′
145S-R:5′-GCGGTGCAGCAACAATACCSNNAATAGAGGCCATATTC-3′
205S-F:5′-GGAAATGAAGGAAGCANNSATTTCAAAACATCCGATGGGAAG-3′
205S-R:5′-CTTCCCATCGGATGTTTTGAAATSNNTGCTTCCTTCATTTCC-3′
PCR condition are as follows: 10 × Buffer, 5 μ L, each 4 μ L, pfu enzyme (2.5U/ of 6 μ L, dNTP (2.5mM) of primer (10mM)
ML) 1 μ L, plasmid 10ng, ultrapure water supply 50 μ L, condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, and 68
DEG C extend 6min, totally 16 circulation.PCR product is with 1 μ LDpnI in 37 DEG C of processing 1h.Then 10 μ L chemical method of PCR product is turned
Enter E.coli DH5a.It send and is sequenced in Shanghai Ying Jun Bioisystech Co., Ltd.After sequencing is correct, extracts plasmid and be transferred to expression bacterium
Strain E.coli BL21-DE3.
The protein expression and purification method and Rate activity measuring method of muton are shown in embodiment 3.1.
Epicycle mutation obtain 9 muton H145A, L205T, L205Q, L205S, L205A, H145A/L205A,
2 mutons H145L, L205M and female parent that H145A/L205T, H145A/L205Q, H145A/L205V and embodiment 2 obtain
The Rate activity compared is shown in Figure of description 1.
The Rate activity of wild type carbonyl reductase ChKRED20 be 17.8 μm of ol/mg/min, muton H145A, L205T,
L205Q, L205S, L205A, H145A/L205A, H145A/L205T, H145A/L205Q, H145A/L205V Rate activity is respectively
118.9,103.7,125.4,96.5,178.3,97.3,123.8,73.2,104.8μmol/mg/min.Wherein, mutant
L205A vigor highest is 10 times of maternal carbonyl reductase ChKRED20.
The maternal optimal reactive temperature and thermal stability with mutant L205A of embodiment 5
The measurement of 5.1 maternal and combination mutant optimal reactive temperatures
Under the conditions of 8.0 pH, the maximum reaction speed of carbonyl reductase wild type and mutant at different temperatures is measured
Rate, reaction temperature are respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C.
Reaction system is (reaction solution of 1mL): the organic phase of 30% volume includes the chloro- 1- of 20mM substrate 2- (3,4- difluorophenyl)
The water phase of ethyl ketone and isopropanol and 70% volume includes the NAD of 0.2g/L+, the kaliumphosphate buffer of 0.1M, pH8.0,
The pure enzyme solution of maternal ChKRED20 of 0.075mg/mL or the pure enzyme solution of L205A of 0.015mg/mL, react 5min at different temperatures.
Isometric ethyl acetate extraction, measures the production quantity of product.Using Rate activity as ordinate, reaction temperature is abscissa, draws enzyme
The curve living changed with reaction temperature.As a result see Figure of description 2.Mutant L205A and female parent all have at 50 DEG C maximum anti-
Rate is answered, and the Rate activity of mutant L205A at different temperatures is all much higher than female parent.
5.2 maternal and mutant L205A thermal stability determinations
The 100 μ L of pure enzyme solution of protein concentration 1mg/mL is placed in the PCR pipe of 250 μ L of capacity, 3 repetitions of each sample are used
Then sample cell is placed in cooled on ice, 4 DEG C, 12000rpm, is centrifuged 10min by the PCR instrument time different in 50 DEG C of processing,
Appropriate treated enzyme solution measurement enzyme activity is taken, measuring method is the same as 3.2.Using the enzyme solution without high-temperature process as reference, obtain
Relative activity.To handle the time as X-axis, the natural logrithm of remnant enzyme activity percentage is Y-axis, does scatterplot with origin75 software
Figure adds Trendline, obtains the linear equation of enzyme heat treatment time and relative activity, the natural logrithm with relative activity 50% is
3.912023 are calculated the corresponding time, and this time is the heat inactivation half-life period t of enzyme1/2.It is maternal under the conditions of 50 DEG C
The t of carbonyl reductase ChKRED201/2For 9.3h, the t of mutant L205A1/2It is mutated for 15.8h to illustrate compared with female parent
The thermal stability of body L205A increases.
Application of 6 mutant of embodiment in the chloro- 1- of biocatalysis 2- (3,4- difluorophenyl) ethyl ketone
Mutant L205A is to replace the 205th leucine residue on the basis of maternal carbonyl reductase ChKRED20
It is changed to alanine residue generation.The optimal reactive temperature of wild type ChKRED20 is 50 DEG C, but stability is not at this temperature
Ideal, therefore real reaction temperature generally uses 40 DEG C.
The concentration of substrate of the reaction system is that the total volume based on reaction calculates gained.Under the conditions of 40 DEG C, 180rpm into
Row reaction.Reaction is 10mL two-phase system: 25% water phase includes 0.1M, the kaliumphosphate buffer of pH 8.0,0.2g/L
NAD+, 3g/L crude enzyme liquid；75% organic phase includes the chloro- 1- of 2- (3,4- difluorophenyl) ethyl ketone 100-200g/L and isopropyl
Alcohol.
The space-time yield of mutant L205A conversion of substrate 2- chloro- 1- (3,4- difluorophenyl) ethyl ketone has very great Cheng than female parent
The raising of degree, the crude enzyme liquid of 3g/L can convert completely the chloro- 1- of 100g/L2- (3,4- difluorophenyl) ethyl ketone in 3h and generate (S)-
Type alcohol, e.e value > 99%, space-time yield 33.3g/ (L.h) are in contrast, maternal then need 16h.As a result see Figure of description 3.
Maternal carbonyl reductase ChKRED20 can at most convert the chloro- 1- of substrate 2- (3,4- difluorophenyl) ethyl ketone that concentration is 150g/L,
And muton L205A can convert the substrate of 200g/L completely in 20h, to illustrate that muton L205A enhances substrate
Tolerance level.
Application of 7 muton of embodiment in biocatalysis 2 '-fluoro acetophenone
The reaction is carried out in 40 DEG C, 180rpm condition, 10mL reaction system are as follows: 25% water phase includes 0.1M, pH
8.0 kaliumphosphate buffer, 0.2g/L NAD+, 3g/L crude enzyme liquid；75% organic phase includes the 2 '-fluoro acetophenones of 20g/L
And isopropanol.Under the conditions of 40 DEG C, the maternal carbonyl reductase ChKRED20 crude enzyme liquid of 3g/L can convert 20g/L's in 12h
2 '-fluoro acetophenones generate R type alcohol, conversion ratio > 99%, e.e value > 99%.Mutant L205A under this condition conversion of substrate when
Empty productivity ratio female parent greatly improves.Muton L205A goes back the 2 '-fluoro acetophenones generation R type alcohol that original content is 20g/L completely and only needs
4h, space-time yield are 3 times of wild type.
Claims (3)
1. a kind of carbonyl reductase ChKRED20 mutant, it is characterised in that with sequence shown in SEQ ID No.2 be to set out sequence
145th Histidine mutagenesis of the enzyme is leucine or alanine by column.
2. carbonyl reductase ChKRED20 mutant described in claim 1, it is characterised in that the 145th histidine of the enzyme
Sport alanine, the 205th leucine sports valine.
3. application of the carbonyl reductase ChKRED20 mutant described in claim 1~2 in catalysis of carbonyl chemical combination object.
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Also Published As
| Publication number | Publication date |
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| CN106047828B (en) | 2019-10-08 |
| CN110016467B (en) | 2021-12-28 |
| CN106047828A (en) | 2016-10-26 |
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