CN102776157B - Improved ketoreductase polypeptide and coding gene thereof, and cell for expressing polypeptide - Google Patents
Improved ketoreductase polypeptide and coding gene thereof, and cell for expressing polypeptide Download PDFInfo
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- CN102776157B CN102776157B CN 201210299300 CN201210299300A CN102776157B CN 102776157 B CN102776157 B CN 102776157B CN 201210299300 CN201210299300 CN 201210299300 CN 201210299300 A CN201210299300 A CN 201210299300A CN 102776157 B CN102776157 B CN 102776157B
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Abstract
The invention relates to an improved ketoreductase polypeptide. The enzymatic activity of the improved ketoreductase polypeptide is higher than that of a wild type ketoreductase polypeptide; the enzymatic activity refers to the enzymatic activity for catalyzing a ketone compound to join in a reduction reaction to generate a chiral alcohol; the wild type ketoreductase polypeptide has an amino acidsequence shown in SEQ ID No. 2 and is from Lactobacillus kefiri DSM 20587; the improved ketoreductase polypeptide contains an amino acid sequence having at least 90% of sequence homology with the amino acid sequence shown in SEQ ID No. 2; and a residue in the amino acid sequence of the improved ketoreductase polypeptide in relation to the 94th residue in the amino acid sequence shown in SEQ ID No. 2 is a tyrosine residue.
Description
Technical field
The cell that the present invention relates to a kind of improved Ketoreductase polypeptides, its encoding gene and express this polypeptide.
Background technology
Synephrine (phenylephrine) has another name called phyenlephrinium, and its chemical formal name used at school is the 3-(2-(N-methylamino)-the 1-hydroxyl) phenol.It is usually used in paroxysmal supraventricular tachycardia clinically, can pass through vasoconstriction, and the rising blood pressure makes the excitement of vagus reflex and decreased heart rate.
The synthetic method of synephrine comprises chemical method and biological process, its ketone substrate Stereoselective reduction is become to have the product of chiral structure.
Adopting biological process to prepare synephrine need use and have stereoselective ketoreductase.
Yet, existing wild-type ketoreductase KRED, its catalytic activity is very low, and for example, the thick enzyme powder of 10g/l can only transform the ketone substrate of 1g/l in 20 hours fully, can't satisfy need of industrial production.
For the biology preparation of synephrine, also there is not a kind of certain stereoselectivity that both had, the ketoreductase that has better catalytic activity again at present.
Summary of the invention
One aspect of the present invention provides a kind of enzymic activity of this improved Ketoreductase polypeptides to be higher than the enzymic activity of wild-type Ketoreductase polypeptides;
Described enzymic activity refers to that catalyzing ketone compound generation reduction reaction generates the enzymic activity of chiral alcohol;
Described wild-type Ketoreductase polypeptides has aminoacid sequence shown in the SEQ ID No.2, and derives from Caucasia lactobacillus Lactobacillus kefiri DSM 20587;
Described improved Ketoreductase polypeptides comprises the aminoacid sequence that at least 90% sequence homology is arranged with aminoacid sequence shown in the SEQ ID No.2, and is tyrosine residues corresponding to the 94th residue in the aminoacid sequence shown in the SEQID No.2 in the aminoacid sequence of this improved Ketoreductase polypeptides.
In a specific embodiments of the present invention, described enzymic activity refers to catalytic substrate 1-(3-hydroxy phenyl)-2-[methyl (phenyl methyl) amino] ethyl ketone generation reduction reaction generates 3-((R)-2-(N-benzyl-N-methylamino)-the 1-hydroxyl) enzymic activity.3-((R)-2-(N-benzyl-N-methylamino)-the 1-hydroxyl) enzymic activity generate synephrine through reduction reaction after sloughing benzyl.
In a specific embodiments of the present invention, described improved Ketoreductase polypeptides has aminoacid sequence shown in SEQ IDNo.4.
In a specific embodiments of the present invention, the enzymic activity of this improved Ketoreductase polypeptides is at least 2 times of enzymic activity of described wild-type Ketoreductase polypeptides.
The present invention also provides the gene of the above-mentioned improved Ketoreductase polypeptides of encoding on the other hand, and it comprises the nucleotide sequence of showing at least 90% sequence homology with nucleotides sequence shown in the SEQ ID No.1.
In a specific embodiments of the present invention, this gene has nucleotide sequence shown in SEQ ID No.3.
Further aspect of the present invention also provides a kind of cell, the improved Ketoreductase polypeptides that this cell expressing is above-mentioned.
Preferably, described cell belongs to lactobacillus or intestinal bacteria.Preferred, described lactobacillus belongs to the Caucasia lactobacillus.In a specific embodiment of the present invention, described intestinal bacteria are E.coli.BL21 (DE3).
Embodiment
To be further elaborated technology contents of the present invention by embodiment below, its objective is content for a better understanding of the present invention.Therefore, for example does not limit protection scope of the present invention.
Embodiment 1
One, preparation can be expressed the host cell of the improved Ketoreductase polypeptides of aminoacid sequence shown in SEQ ID No.4
Step 1: full gene synthesizes nucleotide sequence shown in SEQ ID No.1
Step 2: introduce the saturation mutation to the 94th amino acids
For saturation mutation being introduced nucleotide sequence shown in the SEQ ID No.1, we as the PCR reaction template, and have designed following two pairs of primers with the plasmid pET21b-KREDlk that contains nucleotide sequence shown in the SEQ ID No.1:
Upstream primer F1:5 '-AGGGCTG
CATATGACTGATCGTTTAAAACAC-3 ' (SEQ ID No.5)
Downstream primer R1:5 '-CTC
AAGCTTTTATTGAGCAGTGTATCCACC-3 ' (SEQ ID No.6)
Upstream primer F1 and the acceptance of the bid of downstream primer R1 sequence pour down line place and represent restriction enzyme site:
Mutant primer F2:5 '-CAATGCCGGAAGT
NNSGTCCTCAAGAG-3 ' (SEQ ID No.7)
Mutant primer R2:5 '-CTCTTGAGGAC
SNNACTTCCGGCATTG-3 ' (SEQ IDNo.8)
Mutant primer F2 and the acceptance of the bid of mutant primer R2 sequence pour down the degeneracy base that the mutational site is represented in line place:
Be template with plasmid pET21b-KREDlk, carry out first round PCR reaction, in the following order, each composition is mixed in sterilization EP pipe, adopt 25 μ l reaction system: ddH
2O 16.3 μ l, PCR Buffer 2.5 μ l, dNTP 2 μ l, primers F 1 (10 μ mol/L) 2 μ l, primer R2 (10 μ mol/L) 2 μ l, pET21b-KREDlk 1 μ l, Pfu archaeal dna polymerase (5U/ μ l) 0.2 μ l.Amplification condition is: 94 ℃ of 2min, a circulation; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 20s, 30 circulations; 72 ℃, 10min, a circulation.The amplified production of gained is carried out agarose gel electrophoresis, cut the specific band that glue is recovered in the 300bp place, use vast Tyke dna fragmentation to reclaim test kit fast and reclaim.Carry out second then and take turns the PCR reaction, reaction system is: ddH
2O 16.3 μ l, PCR Buffer 2.5 μ l, dNTP 2 μ l, primers F 2 (10 μ mol/L) 2 μ l, primer R1 (10 μ mol/L) 2 μ l, pET21b-KREDlk 1 μ l, Pfu archaeal dna polymerase 0.2 μ l.Amplification condition is: 94 ℃ of 2min, a circulation; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃, 10min, a circulation.The amplified production of gained is carried out agarose gel electrophoresis, be recovered in the specific band about 500bp.Then, the product of taking turns PCR with the first two is template, carries out third round PCR, and reaction system is as follows: ddH
2O 14.3 μ l, PCR Buffer 2.5 μ l, dNTP 2 μ l, primers F 1 (10 μ mol/L) 2 μ l, primer R1 (10 μ mol/L) 2 μ l, first round PCR reclaims product 1 μ l, and second takes turns PCR reclaims product 1 μ l, Pfu archaeal dna polymerase 0.2 μ l.Amplification condition is: 94 ℃ of 2min, a circulation; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 50s, 30 circulations; 72 ℃, 10min, a circulation.The amplified production of gained is carried out agarose gel electrophoresis, be recovered in the specific band about 800bp.All mutant nucleotide sequences with saturation mutation have then been comprised in this fragment.
Step 3: the expression vector that makes up the sudden change nucleotide sequence
Contain the intestinal bacteria Top10 bacterial strain of plasmid pET21b, overnight incubation in 37 ℃ of shaking tables at the LB inoculation of medium that contains penbritin (Amp, 100 μ g/ml).Get 3ml bacterium liquid and change in the EP pipe, the centrifugal 1min of 12000rpm collects thalline, prepares the operation of test kit specification sheets according to vast Tyke a small amount of plasmid and extracts the pET21b plasmid.
The reaction system of digested plasmid pET21b is: NEB Buffer 25 μ l, and pET21b 43 μ l, NdeI 1 μ l, Hind III 1 μ l is incubated 4h in 37 ℃ of water-baths.Enzyme is cut the gene fragment after the sudden change that step 2 obtains then, adds following reagent successively: NEB Buffer25 μ l, and gene fragment 43 μ l, NdeI 1 μ l, Hind III 1 μ l is incubated 4h in 37 ℃ of water-baths.Add 10 μ l6*loading buffer respectively subsequently in two reaction systems, carry out detected through gel electrophoresis, glue reclaims and is cut into linear dna molecular.
In the EP pipe, add H respectively
2O 3.5 μ l, T4ligase Buffer 1 μ l, pET21b (NdeI, HindIII digested) 2 μ l, step 3 gained fragment is by NdeI, the recovery product 3 μ l that the HindIII enzyme was cut, T4DNA ligase0.5 μ l connects 5h in 16 ℃.
To connect product transformed into escherichia coli BL21 (DE3) bacterial classification: get pipe BL21 (DE3) competent cell (day root biochemical technology company limited), 10 μ l are connected product and the mixing of 100 μ l competent cells, place 30min in the ice-water bath, 42 ℃ of heat shock 90s, place 5min on ice, add LB800 μ l then, in 37 ℃ of shaking tables, cultivate 40min.14,000rpm is centrifugal, abandons 800 μ l supernatants, with the cell that remaining liq suspends and precipitates, coats on the LB flat board that contains 100 μ g/ml Amp, cultivates 12-16h in 37 ℃ of incubators and can obtain transformant.
Then transformant is carried out bacterium colony PCR and identify (use F1, R1 is primer), will contain the 800bp that has an appointment and insert the transformant of fragment as positive colony.Every hole adds LB+Amp substratum 1ml in 96 hole depth orifice plates, selects 94 positive colonies and inoculates into 96 orifice plates, and 16h is cultivated in concussion in 37 ℃ of shaking tables, and this is mutant library.
Step 4: the ketoreductase mutant library that step 3 obtains is induced fermentation
80 μ l bacterium liquid are got in the every hole of the mutant library of overnight incubation inoculated into 96 new orifice plates, the every hole of this plate includes fresh LB+Amp substratum 1ml, and cell grows to OD at 37 ℃ under the 160rpm condition
600Value reaches 0.8 ~ 1.0, add subsequently IPTG to final concentration be 1.0mM, in 30 ℃ of inducing culture 16 hours.Centrifugal, abandon supernatant, collect thalline for detection of active.
Step 5: the host cell (e. coli bl21 (DE3)/pET21b-KRED) of Ketoreductase polypeptides is expressed in screening
Utilize the enzymatic reaction screening to express the host cell of improved Ketoreductase polypeptides.
0.6 the enzymatic reaction system of milliliter, comprising: 10g/L2-(N-benzyl-N-methylamino)-and the 1-(3-hydroxyl) methyl phenyl ketone, 0.1g/L NADPH, 1mM Mg
2+, 500mM buffer solution of sodium phosphate (pH=6.5), the host cell of the centrifugal acquisition of 10g/L step 4.In 30 ℃ of reaction 24h, add equal-volume acetonitrile termination reaction, the centrifugal 5min of 10,000rpm draws on supernatant liquor to the one 96 new orifice plate.Use HPLC to analyze to detect enzyme (method: test column: CNW post alive
Wavelength: 214nm; Moving phase: A phase (H2O+0.1%TFA)-B phase (CH3CN+0.1%TFA), flow velocity is 1.0ml/min, isocratic elution, 23%B+77%A 10.0min stop, 28 ℃ of column temperatures), the area at the product peak that obtains is compared the mutant that the activity that filters out above being of BL21 (DE3)/pET21b-KREDlk activity 30% increases with the reaction peak area of BL21 (DE3)/pET21b-KREDlk zymocyte liquid.
Step 6: identify
In order from the host cell of (the expressing improved Ketoreductase polypeptides) that filter out, to obtain the sequence of ketoreductase, send order-checking with its corresponding bacterial classification, the sequencing result demonstration contains nucleotide sequence shown in the SEQ ID No.3, aminoacid sequence shown in the coding SEQ ID No.4.This bacterial classification called after BL21 (DE3)/pET21b-KREDmu.
Two, the host cell (effect of the e. coli bl21 (DE3)/pET21b-KREDmu) that above-mentioned steps 5 obtains of a kind of improved Ketoreductase polypeptides of the present invention is expressed in checking
With e. coli bl21 (DE3)/pET21b-KREDkl of expressing wild-type Ketoreductase polypeptides (aminoacid sequence shown in the SEQ ID No.2) in contrast, the enzymic activity of the improved Ketoreductase polypeptides of the host cell expression that checking above-mentioned steps 5 obtains.
The 50ml reaction system of preparation R type phenol product:
10g/L 2-(N-benzyl-N-methylamino)-and the 1-(3-hydroxyl) methyl phenyl ketone, 10g/L ketoreductase thalline (e. coli bl21 (DE3)/pET21b-KREDkl or e. coli bl21 (DE3)/pET21b-KREDmu), 10g/L Hexose phosphate dehydrogenase thalline, 200g/L glucose, 0.1g/LNADP
+, 1mM Mg
2+, 50mM buffer solution of sodium phosphate (pH=6.0).Reaction unit utilizes the there-necked flask of 250mL, and 30 ℃ of oil baths utilize magnetic agitation.At first accurately take by weighing 0.5 gram substrate and 10 gram glucose, join in the reaction flask.Add the 40mL deionized water then, 1mM sal epsom, 50mM, the pH6.0 sodium phosphate buffer stirs 10min fast, makes the abundant mixing of reaction system.Measure pH and be about 4.7 this moment, uses the NaOH of 2.5N with pH regulator to 5.1.In system, add 500mg ketoreductase somatic cells, the oxidized form of nicotinamide-adenine dinucleotide I(NADP of 500mg Hexose phosphate dehydrogenase somatic cells and 5mg
+Sodium salt).PH control is between 5.05-5.15 in the entire reaction course, and temperature is controlled at 30 ℃.HPLC analyzes (as described in step 5) behind the reaction 20h.
The HPLC analytical results shows: the improved Ketoreductase polypeptides with aminoacid sequence shown in the SEQ ID No.4 can transform the methyl phenyl ketone substrate fully under screening conditions; And the wild-type ketoreductase with aminoacid sequence shown in the SEQ ID No.2 can only transform 10% less than the methyl phenyl ketone substrate.
Simultaneously, in order to determine the stereoselectivity of mutant, used the lower concentration substrate to react, to guarantee that substrate is converted into product fully, reaction system is: 1g/L 2-(N-benzyl-N-methylamino)-the 1-(3-hydroxyl) methyl phenyl ketone, 0.1g/L NADPH, 1mM Mg
2+, 500mM buffer solution of sodium phosphate (pH=6.5), the BL21 of 10g/L (DE3)/pET21b-KREDkl or BL21 (DE3)/pET21b-KREDmu are in 30 ℃ of reaction 24h.Reaction product is carried out chirality HPLC and is analyzed, the result shows: mutant can be with 2-(N-benzyl-N-methylamino)-the 1-(3-hydroxyl) methyl phenyl ketone is reduced to 3-((R)-2-(N-benzyl-N-methylamino)-the 1-hydroxyl) phenol, its stereoselectivity is not less than the wild-type ketoreductase, the e.e. value〉99.9%.
SEQUENCE LISTING
＜110〉section's biological medicine (Shanghai) Co., Ltd. still
＜120〉improved Ketoreductase polypeptides, its encoding gene and the cell of expressing this polypeptide
<130> 2012hj3
<160> 8
<170> PatentIn version 3.5
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＜213〉Caucasia lactobacillus Lactobacillus kefiri DSM 20587
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＜213〉Caucasia lactobacillus Lactobacillus kefiri DSM 20587
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＜213〉Caucasia lactobacillus Lactobacillus kefiri DSM 20587
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Claims (9)
1. an improved Ketoreductase polypeptides is characterized in that,
The enzymic activity of this improved Ketoreductase polypeptides is higher than the enzymic activity of wild-type Ketoreductase polypeptides;
Described enzymic activity refers to that catalyzing ketone compound generation reduction reaction generates the enzymic activity of chiral alcohol;
The aminoacid sequence of described wild-type Ketoreductase polypeptides and derives from Caucasia lactobacillus Lactobacillus kefiri DSM20587 shown in SEQ ID No.2;
Be tyrosine residues corresponding to the 94th residue in the aminoacid sequence shown in the SEQ ID No.2 in the aminoacid sequence of described improved Ketoreductase polypeptides, the part in its aminoacid sequence except described the 94th residue is identical with sequence shown in the SEQ ID No.2.
2. improved Ketoreductase polypeptides as claimed in claim 1, it is characterized in that: described enzymic activity refers to catalytic substrate 1-(3-hydroxy phenyl)-2-[methyl (phenyl methyl) amino] ethyl ketone generation reduction reaction generates 3-((R)-2-(N-benzyl-N-methylamino)-the 1-hydroxyl) enzymic activity.
3. improved Ketoreductase polypeptides as claimed in claim 1, it is characterized in that: the aminoacid sequence of described improved Ketoreductase polypeptides is shown in SEQ ID No.4.
4. improved Ketoreductase polypeptides as claimed in claim 1 is characterized in that: the enzymic activity of this improved Ketoreductase polypeptides is at least 2 times of enzymic activity of described wild-type Ketoreductase polypeptides.
5. a coding is characterized in that as the gene of improved Ketoreductase polypeptides as described among the claim 1-4 any:
Encode the nucleotide sequence of gene of described improved Ketoreductase polypeptides shown in SEQ ID No.3.
6. a cell is characterized in that: any described improved Ketoreductase polypeptides among this cell expressing claim 1-4.
7. as cell as described in the claim 6, it is characterized in that: described cell belongs to lactobacillus or intestinal bacteria.
8. as cell as described in the claim 7, it is characterized in that: described lactobacillus belongs to the Caucasia lactobacillus.
9. as cell as described in the claim 7, it is characterized in that: described intestinal bacteria are E.coli.BL21 (DE3).
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| CN105985990B (en) * | 2015-02-03 | 2020-01-10 | 苏州汉酶生物技术有限公司 | Production method of phenylephrine intermediate |
| CN107326049A (en) * | 2016-04-28 | 2017-11-07 | 尚科生物医药（上海）有限公司 | Recombinate ketoreductase and prepare (R) -3,5- two（Trifluoromethyl）Application in benzyl carbinol |
| CN106011095B (en) * | 2016-07-27 | 2021-02-26 | 苏州汉酶生物技术有限公司 | Engineered ketoreductase polypeptide and method for preparing ezetimibe intermediate by using same |
| CN109423484B (en) * | 2017-09-04 | 2021-06-25 | 尚科生物医药（上海）有限公司 | Ketoreductase and application thereof in preparation of (S) -2-chloro-1- (3, 4-difluorophenyl) ethanol |
| CN109576313B (en) * | 2017-09-29 | 2022-02-22 | 尚科生物医药（上海）有限公司 | Method for preparing (S) -2-chloro-1- (3-hydroxyphenyl) ethanol |
| CN109456949A (en) * | 2018-12-27 | 2019-03-12 | 尚科生物医药（上海）有限公司 | A kind of Ketoreductase mutant being used to prepare R type neo-synephrine |
| CN110938608A (en) * | 2019-12-20 | 2020-03-31 | 台州酶易生物技术有限公司 | Aldehyde ketone reductase mutant, encoding gene and application of aldehyde ketone reductase mutant in synthesis of (S) -TCPE |
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|---|---|---|---|---|
| WO2008103248A1 (en) * | 2007-02-08 | 2008-08-28 | Codexis, Inc. | Ketoreductases and uses thereof |
| WO2008122511A2 (en) * | 2007-04-04 | 2008-10-16 | F. Hoffmann-La Roche Ag | Process for preparing 2-amino- [5-(1(s), 2-dihydroxyethyl) or - (ks) -hydroxy-2-haloethyl) ] -pyrazine derivatives by enzymatic reduction of the corresponding ketones |
| ATE547513T1 (en) * | 2007-09-28 | 2012-03-15 | Codexis Inc | KETOREDUCTASE POLYPEPTIDES AND USES THEREOF |
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