CN101724597B - Method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by mRNA two-stage structure - Google Patents
Method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by mRNA two-stage structure Download PDFInfo
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- CN101724597B CN101724597B CN2009102631464A CN200910263146A CN101724597B CN 101724597 B CN101724597 B CN 101724597B CN 2009102631464 A CN2009102631464 A CN 2009102631464A CN 200910263146 A CN200910263146 A CN 200910263146A CN 101724597 B CN101724597 B CN 101724597B
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Abstract
The invention relates to a method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by an mRNA two-stage structure, belonging to the technical field of biocatalysis asymmetric transformation. The invention optimizes an mRNA translation start area two-stage structure of (R)-carbonyl reductase and constructs a corresponding mutation recombination strain which is classified and named Escherichia coli BL21/pET32a-mrcr with the preservation CCTCC No. M209289. The mutation strain not only realizes the efficient expression of the (R)-carbonyl reductase but also effectively improves the zymoprotein activity and biotransformation efficiency, and provides novel research idea and approach for efficiently preparing the (R)-phenylglycol in high substrate concentration.
Description
Technical field
The method of the bio-transformation efficient of mRNA secondary structure optimization and improvement (R)-carbonyl reductase, by optimizing the secondary structure in mRNA translation initiation district, overcome sterically hindered that protein translation starts, promote efficiently expressing of target protein and correctly folding of space structure thereof, effectively improve the method and the application of zymoprotein vigor and biocatalytic Activity, belong to the asymmetric transformation technology of biocatalysis field.
Background technology
Chiral alcohol has special photoelectromagnetism performance and physiologically active, is very ideal chiral drug intermediate and liquid crystal material adulterating agent.Optical purity phenylglycol particularly, a kind of important chirality module compound in medicine, agricultural chemicals and the chemical materials especially.Utilize reorganization oxydo-reductase asymmetric reduction prochiral ketone to prepare the optics chiral alcohol, become the main development trend of current industrial production chiral alcohol.
The initial process of mRNA translation initiation region two-stage stability of structure and target protein translation is closely related.According to statistics, the every increase of Gibbs free energy (Δ G) absolute value 1.4kcal/mol in the mRNA translation initiation region two-stage structure, translation initiation efficient will reduce by 10 times.
The phenylglycol of Candida parapsilosis cell catalysis DL can obtain optically active (S)-phenylglycol, and the key enzyme that participates in reaction has two kinds, i.e. (R)-carbonyl reductase and (S)-carbonyl reductase.Reversible reaction between the former catalysis (R)-phenylglycol and the 2-hydroxy acetophenone; The latter is a substrate with intermediate 2-hydroxy acetophenone, produces (S)-phenylglycol; Because (R)-expression level of carbonyl reductase and catalytic efficiency be well below (S)-carbonyl reductase, causes Candida parapsilosis can only synthesize (S)-phenylglycol.On this research basis, this laboratory with in the mRNA translation initiation district+1～+ 78 district is as optimizing object, a plurality of Nucleotide are implemented rite-directed mutagenesis, make up the corresponding mutant of optimizing, realized efficiently expressing of (R)-carbonyl reductase, enzyme activity and bio-transformation efficient have been improved, for optically active (the R)-phenylglycol of efficient production provides brand-new route.
Summary of the invention
1, the technical problem that will solve.The purpose of this invention is to provide a kind of mRNA translation initiation region two-stage composition optimizes technology and improve the expression level and the catalysis of enzyme, utilize the method for efficient asymmetric conversion preparation (the R)-phenylglycol of recombinant bacterial strain (culture presevation number: CCTCCNO:M 209289).The object of the invention is by 14 nucleotide sites in the secondary structure in (R)-carbonyl reductase mRNA translation initiation district are implemented same sense mutation, make the enzyme protein expression level improve 4～5 times, enzyme activity improves 61.9%, high density (5.0g/L) substrate 2-hydroxy acetophenone is carried out catalyzed reaction, the optical purity and the productive rate of product (R)-phenylglycol reach 95.2%e.e. and 83.3% respectively, have improved 30.4% and 43.1% before optimizing.For having optically active (R)-phenylglycol, efficient production provides novel research thinking and reference.
2, technical scheme:
(1), the reorganization bacterium of the asymmetric conversion preparation of a strain (R)-phenylglycol, its classification called after intestinal bacteria (Escherichia coli) BL21/pET32a-mrcr, be preserved in Chinese typical culture collection center, deposit number: CCTCC NO:M209289.
(2), the construction process of described reorganization bacterium CCTCC NO:M209289, mutator gene mrcr is inserted carrier pET32a construction recombination plasmid pET32a-mrcr, recombinant plasmid pET32a-mrcr transformed into escherichia coli E.coli BL21 (DE3) competent cell, by containing the LB plate screening of 100 μ g/mL penbritins, obtain recombinant bacterial strain E.coli BL21/pET32a-mrcr, i.e. CCTCC NO:M209289; Step is:
1. the optimization of gene: according to the MFOLD software algorithm, to part Nucleotide in the mRNA translation initiation district of (R)-carbonyl reduction enzyme coding gene rcr (+1nt～+ 78nt) analyze, stability or free energy Δ G with the reduction secondary structure are foundation, and 13 Nucleotide have been carried out rite-directed mutagenesis; By A:(+1) atgtca att cca tca agc cag tac gga ttc gta ttc aat aag caa tca gga ctt aag ttg aga aat gat ttgcct gt
2. the acquisition of mrcr: with recombinant plasmid pETRCR as the PCR reaction template, utilization contains the primer mRCR_F of NcoI restriction enzyme site, contains the primer mRCR_R of XhoI restriction enzyme site, passes through pcr amplification reaction, obtain the mrcr gene, its total length is 1008bp;
mRCR_F：5’
-GACCATGGGATCAATACCATCATCACAATACGGATTCGTATTCAATAAGCAATCAGGATTAAAACTACGTAACGATTTG-3’，
mRCR_R：5’-TGACTCTCGAGTGGATTAAAAACAACTCTACCTTCATAAGCATTGTT-3’，
PCR reaction system: ddH
2O 37.5 μ L, 10 * Reaction Buffer, 5 μ L, the dNTP4 μ L of 25mmol/L, each 1 μ L of the primer mRCR_F of 50pmol/ μ L and mRCR_R, recombinant plasmid pETRCR 1 μ L, the Taq DNA polymerase 0.5 μ L of 5U/ μ L;
PCR reaction conditions: 95 ℃ of pre-sex change 5min; 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; Obtain the mrcr gene;
3. the structure of recombinant plasmid pET32a-mrcr: utilize restriction enzyme NcoI and XhoI that gene mrcr and carrier pET32a are carried out double digestion respectively earlier and handle, handle the back dna segment and connect, obtain to have the recombinant plasmid pET32a-mrcr of gene mrcr by sticky end;
4. recombinant plasmid transformed intestinal bacteria: get recombinant plasmid pET32a-mrcr 2 μ L, transformed into escherichia coli E.coli BL21 (DE3) competent cell, conversion fluid is applied on the LB flat board that contains 100 μ g/mL penbritins, be inverted overnight incubation for 37 ℃, obtain positive colony E.coli BL21/pET32a-mrcr.
(3), utilize the asymmetric conversion preparation of the reorganization bacterium CCTCC NO:M209289 that makes up behind the rite-directed mutagenesis (R)-phenylglycol:
(a) cultivation of recombinant bacterial strain CCTCC NO:M209289
Adopt the LB liquid nutrient medium in g/L: Tryptones 10, yeast extract 5, NaCl 10, and pH 7.0; Solid medium adds the agar powder of liquid nutrient medium weight 1.5% again;
Culture condition: single colony inoculation of picking recombinant bacterial strain CCTCC NO:M209289 contains in the LB liquid nutrient medium of 100 μ g/mL penbritins in 3mL, spends the night in 37 ℃ of 200rpm shaking culture; Get the 1mL nutrient solution and transfer and contain in the LB liquid nutrient medium of 100 μ g/mL penbritins in 50mL, in 37 ℃ of 200rpm shaking culture to OD
600Be 0.6～0.8; Add inductor isopropyl-1mmol/L, 30 ℃ of following inducing culture spend the night, and 10, the centrifugal 10min of 000rpm collects thalline, uses the physiological saline washed twice, collects the full cell of bacterium that obtains recombinating;
(b) be catalyzer with the full cell of reorganization bacterium, with the 2-hydroxy acetophenone is substrate, carry out asymmetric conversion reaction: the acetate buffer solution of 5mL 0.1mol/L pH 5.0～6.0, or the phosphoric acid buffer of 5mL 0.1mol/L pH 7.0, or in the Tris-HCl damping fluid of 5mL 0.1mol/L pH 8.0～9.0, the full cell concn of reorganization bacterium is 0.1～0.2g/mL, and concentration of substrate is 5g/L, 30 ℃ of temperature of reaction, the reaction times is 48h.
After reaction finished, with the centrifugal thalline of removing of reaction mixture, supernatant liquor added 2 times of volumes of acetic acid ethyl ester extractions, and organic phase is used for analyzing; Product is analyzed by chiral stationary phase high performance liquid chromatography (Agillent HP1100), and condition is Chiralcel OB-H post (4.6mm * 25cm; Daicel Chemical Ind., Ltd., Japan), moving phase be normal hexane/Virahol (9/1, V/V), flow velocity 0.5mL/min, the detection wavelength is 215nm.The optical purity of product is weighed by the mapping excessive value.
The calculating of product (R)-phenylglycol mapping excessive value:
Mapping excessive value (e.e.%)=[(C
R-C
S)/(C
R+ C
S)] * 100%
The calculating of product (R)-phenylglycol productive rate: productive rate (%)=C
R/ C
0* 100%
C in the formula
RFor reacting the concentration of back (R)-phenylglycol, C
SFor reacting the concentration of back (S)-enantiomorph, C
0For reacting the concentration of preceding substrate (R)-phenylglycol.
The full cell of reorganization bacterium is a catalyzer, and behind the asymmetric bioconversion reaction, product is (R)-phenylglycol.
3, beneficial effect: be optimized by mRNA translation initiation region two-stage structure to (R)-carbonyl reductase (the GenBank registration number DQ675534 of encoding gene), utilize the reorganization bacterium E.coli BL21/pET32a-mrcr that successfully makes up to carry out bio-transformation 2-hydroxy acetophenone, obtain product (R)-phenylglycol.By optimizing reaction conditions, in the Tris-HCl of pH8.5 damping fluid, utilize 0.2g/mL to recombinate full cell to 5g/L substrate 2-hydroxy acetophenone catalyzed conversion 48h, the optical purity of final product (R)-phenylglycol is 95.2%e.e., productive rate is 83.3%.These work provide effective way for the efficient conversion of (R)-phenylglycol, and the catalysis of the optimization and improvement biological enzyme by mRNA translation initiation region two-stage structure provides research thinking and reference.
The biological material specimens preservation
Intestinal bacteria (Escherichia coli) BL21/pET32a-mrcr, depositary institution: Chinese typical culture collection center, be called for short CCTCC, deposit number: CCTCC NO:M 209289, preservation date: on November 29th, 2009.
Description of drawings
The contrast of nucleotide sequence and translation initiation region two-stage structure before and after Fig. 1 optimizes.
(A): use the mRNA translation initiation district (from+1 to+78) of (the R)-carbonyl reductase of MFOLD method prediction to optimize preceding secondary structure; (B): the secondary structure after the mRNA translation initiation district (from+1 to+78) of (the R)-carbonyl reductase of usefulness MFOLD method prediction optimizes.
Embodiment
The optimization of embodiment 1 gene:
With the stability or the free energy (Δ G) that reduce secondary structure is foundation, to 13 Nucleotide in the mRNA translation initiation district of (R)-carbonyl reduction enzyme coding gene rcr, and promptly the 9th, 16,17,18,21,52,54,57,58,60,61,63 and 66 t, a, g, c, c, c, t, g, t, g, a, a, t sports A, T, C, A, A, T, A, A, C, A, C, T, C.
The acquisition of embodiment 2mrcr gene:
As the PCR reaction template, utilize mRCR_F that contains the NcoI restriction enzyme site and the mRCR_R that contains the XhoI restriction enzyme site to make primer with recombinant plasmid pETRCR:
mRCR_F：5’-GACCATGGGATCAATACCATCATCACAATACGGATTCGTATTCAATAAGCAATCAGGATTAAAACTACGTAACGATTTG-3’，
mRCR_R：5’-TGACTCTCGAGTGGATTAAAAACAACTCTACCTTCATAAGCATTGTT-3’，
PCR reaction system: ddH
2O 37.5 μ L, 10 * Reaction Buffer, 5 μ L, 25mmol/L dNTP4 μ L, each 1 μ L of the primer mRCR_F of 50pmol/ μ L and mRCR_R, recombinant plasmid pETRCR 1 μ L, 5U/ μ L Taq DNA polymerase 0.5 μ L; PCR reaction conditions: 95 ℃ of 5min; 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; The PCR reaction obtains the mrcr gene.
The acquisition of embodiment 3 recombinant plasmid pET32a-mrcr:
Utilize restriction enzyme NcoI and XhoI that gene mrcr and pET32a (the pET32a plasmid derives from Novagen company) are carried out the double digestion processing respectively, handle the back dna segment and connect, obtain to have the recombinant plasmid pET32a-mrcr of gene mrcr by sticky end.Utilize plasmid extraction kit Mini-PlasmidRapid Isolation Kit (buy in Beijing vast Tyke biological gene technology company limited) to extract plasmid pET32a-mrcr.
The acquisition of embodiment 4 recombinant bacterial strain E.coli BL21/pET32a-mrcr:
Recombinant plasmid transformed intestinal bacteria: in 100 μ L E.coli BL21 (DE3) competent cell suspensions of every pipe, add 10 μ L and connect product, ice bath 30min behind the mixing gently.Change 42 ℃ of water-baths over to, thermal shock 90s.Be transferred in the ice bath cooling 2min fast.Add 700 μ L LB liquid nutrient mediums in every pipe, 37 ℃ of 100rpm shaking table incubations are cultivated 1h.Cultivate back bacterium liquid 3, the centrifugal 2min of 000rpm abandons supernatant 600 μ L, is coated with behind the residue bacterium liquid mixing to contain on the LB flat board of 100 μ g/mL penbritins, is inverted overnight incubation for 37 ℃.
The LB substratum: Tryptones 1%, yeast extract 0.5%, NaCl 1%, and pH 7.0.Add penbritin (100 μ g/mL) before using, solid medium adds 1.5% agar powder.
4 clones of picking, switching goes into to be equipped with in the LB liquid nutrient medium that contains 100 μ g/mL penbritins of 3mL, cultivate 12h for 37 ℃, utilize plasmid extraction kit Mini-Plasmid Rapid Isolation Kit (vast Tyke, Beijing biological gene technology company limited) from the bacterium liquid of cultivating, to extract plasmid.Cut the system checking through following enzyme: 10 * Buffer H, 2 μ L, plasmid DNA 5 μ L, Nco I 0.5 μ L, XhoI 0.5 μ L, ddH
2O supplies 20 μ L with system.Enzyme is cut the positive thalline of result and is reorganization bacterium E.coli BL21/pET32a-mrcr (bacterium is hidden numbering CCTCC NO:M209289).
The cultivation of embodiment 5 reorganization bacterium:
The LB liquid nutrient medium is as described in the specification sheets.Single colony inoculation of reorganization bacterium E.coliBL21/pET32a-mrcr contains in the LB liquid nutrient medium of 100 μ g/mL penbritins in 3mL among the picking embodiment 3, in 37 ℃, the 200rpm shaking culture is spent the night, getting the 1mL nutrient solution transfers and contains in the LB liquid nutrient medium of 100 μ g/mL penbritins in 50mL, in 37 ℃, the 200rpm shaking culture is to OD
600After being 0.6～0.8, add the 1mmol/L isopropyl-, spend the night in 30 ℃ of inducing culture; 10, the centrifugal 10min of 000rpm collects thalline, with physiological saline washing thalline twice, collects the full cell of reorganization bacterium.
The expression of embodiment 6 target proteins
With the bacterium liquid among the embodiment 5, carry out SDS-PAGE and detect, gel quantitation software (Quantity One) analyzing proteins is expressed; The reorganization bacterium of collecting is dissolved in 20mmol/L, in the Tris-HCl damping fluid of pH8.0, ultrasonication 20min, working hour 2s, intermittent time 6s.In 16, the centrifugal 40min of 000rpm gets cleer and peaceful precipitation respectively with the thalline after the fragmentation, and the SDS-PAGE detected result shows that (R)-carbonyl reductase expressing quantity is 4～5 times before optimizing.
Embodiment 7
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L acetate buffer solution (pH 5.0), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and product is 28.5%e.e. for the optical purity of (R)-phenylglycol, and productive rate is 16.9%.
Embodiment 8
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L acetate buffer solution (pH 6.0), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and product is 43.7%e.e. for the optical purity of (R)-phenylglycol, and productive rate is 32.6%.
Embodiment 9
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L phosphoric acid buffer (pH 7.0), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and the optical purity of product (R)-phenylglycol is 74.5%e.e., and productive rate is 58.2%.
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L Tris-HCl damping fluid (pH 8.0), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, the optical purity 90.7%e.e. of product (R)-phenylglycol, productive rate 80.4%.
Embodiment 11
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L Tris-HCl damping fluid (pH8.5), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and the optical purity of product (R)-phenylglycol is 93.1%e.e., and productive rate is 81.8%.
Embodiment 12
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L Tris-HCl damping fluid (pH9.0), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.1g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and the optical purity of product (R)-phenylglycol is 89.6%e.e., and productive rate is 78.9%.
Embodiment 13
Carry out the bio-transformation test with the thalline that is obtained among the embodiment 5.In 5mL 0.1mol/L Tris-HCl damping fluid (pH8.5), add 5g/L substrate 2-hydroxy acetophenone respectively, 0.2g/mL recombination bacillus coli wet thallus, behind the mixing on 30 ℃ of constant temperature shaking tables oscillatory reaction 48h.Reaction back mixture is centrifugal, gets the supernatant liquor extraction, and the optical purity of product (R)-phenylglycol is 95.2%e.e., and productive rate is 83.3%.
Claims (2)
1. the reorganization bacterium of the asymmetric conversion of strain preparation (R)-phenylglycol, its classification called after intestinal bacteria (Escherichia coli) BL21/pET32a-mrcr, be preserved in Chinese typical culture collection center, deposit number: CCTCC NO:M209289.
2. utilize the method for the asymmetric conversion preparation of the reorganization bacterium CCTCC NO:M209289 that makes up behind the rite-directed mutagenesis (R)-phenylglycol, it is characterized in that step is:
(1) cultivation of recombinant bacterial strain CCTCC NO:M209289
Adopt the LB liquid nutrient medium in g/L: Tryptones 10, yeast extract 5, NaCl 10, and pH 7.0; Solid medium adds the agar powder of liquid nutrient medium weight 1.5% again;
Culture condition: single colony inoculation of picking recombinant bacterial strain CCTCC NO:M209289 contains in the LB liquid nutrient medium of 100 μ g/mL penbritins in 3mL, spends the night in 37 ℃ of 200rpm shaking culture; Get the 1mL nutrient solution and transfer and contain in the LB liquid nutrient medium of 100 μ g/mL penbritins in 50mL, in 37 ℃ of 200rpm shaking culture to OD
600Be 0.6～0.8; Add inductor isopropyl-1mmol/L, 30 ℃ of following inducing culture spend the night, and 10, the centrifugal 10min of 000rpm collects thalline, uses the physiological saline washed twice, collects the full cell of bacterium that obtains recombinating;
(2) be catalyzer with the full cell of reorganization bacterium, with the 2-hydroxy acetophenone is substrate, carry out asymmetric conversion reaction: the acetate buffer solution of 5mL 0.1mol/L pH 5.0～6.0, or the phosphoric acid buffer of 5mL 0.1mol/L pH 7.0, or in the Tris-HCl damping fluid of 5mL 0.1mol/L pH 8.0～9.0, the full cell concn of reorganization bacterium is 0.1～0.2g/mL, and concentration of substrate is 5g/L, 30 ℃ of temperature of reaction, the reaction times is 48h.
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Address after: 214122 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: 214122 Jiangsu Province, Wuxi City Lake Road No. 1800, Jiangnan University Institute of biological engineering Patentee before: Jiangnan University |