CN101870984B - RSP_2728 esterase mutant genes obtained by directed evolution and application of esterase in methyl mandelate resolution reaction - Google Patents

RSP_2728 esterase mutant genes obtained by directed evolution and application of esterase in methyl mandelate resolution reaction Download PDF

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CN101870984B
CN101870984B CN2010101868653A CN201010186865A CN101870984B CN 101870984 B CN101870984 B CN 101870984B CN 2010101868653 A CN2010101868653 A CN 2010101868653A CN 201010186865 A CN201010186865 A CN 201010186865A CN 101870984 B CN101870984 B CN 101870984B
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esterase
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ala
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CN101870984A (en
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于洪巍
汤晓玲
刘击
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Zhejiang University ZJU
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Abstract

本发明属于基因工程与酶工程领域。公开了酯酶基因RSP-2728的突变基因及其基因工程的应用。通过定向进化手段，采用易错PCR和基因克隆方法，以及一种适用于活性以及立体选择性的高通量筛选方法，通过四轮筛选，获得了最终突变酯酶XLA，XLB，XLC和XLD。将其应用于扁桃酸甲酯的拆分反应中，证实立体选择性最高的突变酯酶XLD其立体选择性与野生型相比较，其对映体选择性(E值)从E＝3.1提高到了E＝30.8；对底物的反应活性最高的XLB与野生型相比较，活性从10U/mg，最高提高到了142U/mg。在酯酶拆分消旋底物或者合成重要的药物中间体中，具有较好的应用前景。The invention belongs to the fields of genetic engineering and enzyme engineering. Disclosed is the mutant gene of the esterase gene RSP-2728 and its application in genetic engineering. By means of directed evolution, using error-prone PCR and gene cloning methods, and a high-throughput screening method suitable for activity and stereoselectivity, the final mutant esterases XLA, XLB, XLC and XLD were obtained through four rounds of screening. It was applied to the resolution reaction of methyl mandelate, and it was confirmed that the stereoselectivity of the mutant esterase XLD with the highest stereoselectivity was compared with that of the wild type, and its enantioselectivity (E value) was improved from E=3.1 to E=30.8; compared with the wild type, the activity of XLB, which has the highest reaction activity to the substrate, increased from 10U/mg to 142U/mg. It has a good application prospect in the separation of racemic substrates by esterases or the synthesis of important drug intermediates.

Description

RSP_2728 esterase mutant genes and the application of esterase in methyl mandelate resolution reaction through the acquisition of orthogenesis means
Technical field
The invention belongs to enzyme engineering and genetically engineered field; Relate in particular to the two mutants that obtains the esterase RSP_2728 gene (GeneID:3720458) of the red bacterium of type of coming from ball (Rhodobacter sphaeroides) bacterial strain through the orthogenesis means, and with mutator gene expression and the application in intestinal bacteria afterwards that be connected with expression vector pET-30a (+).
Background technology
Esterase is one type of crucial biological catalyst; It can also generate corresponding acid and pure by a series of ester bonds of hydrolyzation catalysis; Its stereoselectivity is a key property of its catalyzed reaction, is widely used in important chipal compounds such as synthesised food additive, agrochemicals and medicine intermediate.Enzymatic stereoselectivity reaction is one of focus of current chiral drug study on the synthesis.
The enzyme that obtains from occurring in nature can not reach the desired level of chiral separation, often has certain limitation, and therefore, the stereoselectivity that improves enzyme through correlation technique has great importance.Under the situation to enzymatic structure or catalytic mechanism poor information, the orthogenesis technology provides strong instrument for the new biocatalysis of exploitation.Many researchs show, adopt the orthogenesis technology can improve the stereoselectivity of enzyme.
Summary of the invention
The purpose of this invention is to provide a kind of RSP_2728 esterase mutant genes and application thereof that obtains through the orthogenesis means.
The present invention also aims to provide a kind of esterase and application thereof of encoding through the RSP_2728 esterase mutant genes of orthogenesis means acquisition.
The mutator gene of the RSP_2728 gene of the red bacterium of one kind ball (Rhodobacter sphaeroides) is characterized in that the nucleotide sequence of said mutator gene is one of following nucleotide sequences:
(1) nucleotide sequence of SEQ ID NO:1 in the sequence table;
(2) nucleotide sequence of SEQ ID NO:2 in the sequence table;
(3) nucleotide sequence of SEQ ID NO:3 in the sequence table;
(4) nucleotide sequence of SEQ ID NO:4 in the sequence table;
By the esterase of mutator gene coding, the aminoacid sequence of said esterase is one of following amino acid sequences:
(1) aminoacid sequence of SEQ ID NO:5 in the sequence table;
(2) aminoacid sequence of SEQ ID NO:6 in the sequence table;
(3) aminoacid sequence of SEQ ID NO:7 in the sequence table;
(4) aminoacid sequence of SEQ ID NO:8 in the sequence table;
Wherein, the amino acid sequences encoded esterase called after XLA of SEQ ID NO:5 in the sequence table becomes tyrosine with respect to its asparagine mutation of the 62nd of wild-type; The amino acid sequences encoded esterase called after XLB of SEQ ID NO:6 in the sequence table becomes tyrosine, the 145th leucine to be mutated into Histidine with respect to its asparagine mutation of the 62nd of wild-type; The amino acid sequences encoded esterase called after XLC of SEQ ID NO:7 in the sequence table becomes halfcystine, the 145th leucine to be mutated into Histidine with respect to its 62nd asparagine mutation of wild-type; The amino acid sequences encoded esterase called after XLD of SEQ ID NO:8 in the sequence table becomes with respect to its 62nd asparagine mutation of wild-type that halfcystine, the 121st methionine(Met) are mutated into a word used in person's names propylhomoserin, the 145th leucine is mutated into Histidine.
The preparation method of esterase XLA, XLB, XLC or XLD; It is characterized in that: according to the design of the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:4 primer; Utilize the method for pcr amplification to obtain amplified production and with it with after expression vector pET-30a (+) links to each other; Be transformed in the e. coli bl21 host cell, behind abduction delivering, obtain the reorganization esterase.
The application of the XLA of esterase, XLB, XLC or XLD; It is characterized in that: the resolution reaction that said esterase is used for methyl mandelate; When being esterase XLA, from wild-type, enantio-selectivity has been brought up to E=5.3 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it; It from wild-type, has brought up to 43U/mg from 10U/mg to the activity of methyl mandelate; When being esterase XLB, from wild-type, enantio-selectivity has been brought up to E=8.8 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it, and it from wild-type, has brought up to 142U/mg from 6U/mg to the activity of methyl mandelate; When being esterase XLC, from wild-type, enantio-selectivity has been brought up to E=14.0 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it, and it from wild-type, has brought up to 88U/mg from 6U/mg to the activity of methyl mandelate; When being esterase XLD, from wild-type, enantio-selectivity has been brought up to E=30.8 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it, and it from wild-type, has brought up to 20U/mg from 6U/mg to the activity of methyl mandelate.
A kind of transformed host cell that contains esterase RSP_2728 mutator gene, it is that the recombinant vectors that contains esterase gene is imported to the host; Said host is a bacillus coli DH 5 alpha, one of BL21 or JM109.
A kind of is indicator with bromine Thymus vulgaris Finland, confirms the high-throughput screening method that enzyme is lived through the monitoring Sorensen value that ester hydrolysis reaction produced.
The RSP_2728 wild type gene of the class red bacterium of ball (Rhodobacter sphaeroides) bacterial strain, its nucleotides sequence is classified the nucleotide sequence shown in the SEQ ID NO:11 in the sequence table as.
The present invention is through carrying out genetic engineering modified to the esterase RSP_2728 in a red bacterium of class ball (Rhodobacter sphaeroides) bacterial strain; Study from molecular level; Obtained in the resolution reaction process of racemic ester; The mutant strain that the enzyme stereoselectivity obviously improves is applicable to industrial application.
The sudden change esterase XLA that is obtained among the present invention; XLB, XLC, the characteristics of XLD are to compare with the esterase of wild-type; When splitting the racemic mandelic acid methyl esters; Its stereoselectivity tool is significantly improved, and the stereoselectivity of XLD reaches the highest: E=30.8, and its vigor also is significantly improved than wild-type; XLB is the highest to the reaction vigor of substrate, reaches 142U/mg.
Description of drawings:
Fig. 1 has shown a kind of construction strategy figure that contains the recombinant expression vector of esterase gene.
Embodiment
The molecular cloning and the expression of embodiment 1 esterase RSP_2728 mutator gene
1, obtains esterase mutant genes through fallibility PCR
Utilize the RSP_2728 gene in the red bacterium of fallibility pcr amplification class ball (Rhodobacter sphaeroides) bacterial strain (obtain from Chinese common micro-organisms preservation administrative center, culture presevation is numbered CGMCC 1.1737), obtain mutant nucleotide sequence.In the fallibility pcr amplification reaction, amplimer is S2a (5 '-CGGGATCCATGACACGCAAGCTGACGTTCG-3 ') and S2b (5 '-CCCAAGCTTTCAGTCCGGCAGACGCTCCTTC-3 ').
Amplification system is: 50 μ l reaction systems:
10x fallibility PCR damping fluid: 3-8 μ l;
Mg 2+(25mM/L)：2-8μl；
DNTP (10mmol/L dGTP, 10mmol/L dATP, 10mmol/L dCTP and 10mmol/LdTTP mixture): 0.5-3 μ l;
Primer S2a and S2b:5-40pmol;
Dna profiling: 5-40pmol;
Taq archaeal dna polymerase: 5-10U;
Supply system with distilled water.
Amplification condition: 95 ℃ of 2-3min; 95 ℃ of 45-60s, 50-59 ℃ of 40-90s, 72 ℃ of 40-90s, 30-35 circulation; 72 ℃ of 10min.
The final fallibility pcr amplification product that obtains is used to make up esterase sudden change library.
2, the structure in esterase sudden change library
With the agarose gel electrophoresis of fallibility pcr amplification product, carry out purifying and recovering with PCR purifying and recovering test kit with 1% (W/V).Fallibility pcr amplification product behind the purifying with corresponding restriction enzyme carry out 37 ℃ of enzymes cut spend the night the back be connected with pET-30a (+); The super competent cell of transformed into escherichia coli BL21; Coat on LB (containing 50 μ g/ml kantlex) the solid culture plate; 37 ℃ of cultivations are selected to have and are inserted segmental clone, and the clone of acquisition is built into into mutant library.
Active and the stereoselective screening of embodiment 2 esterase RSP_2728 mutator genes
1, the high-throughput abduction delivering of two mutants
Adopt 96 microwell plates to express.Single bacterium colony on the LB solid culture plate selected one by one contain in 200 μ l/ hole LB substratum, 96 orifice plates of (containing 50 μ g/ml kantlex) (this is called motherboard below plate), with motherboard overnight cultures in 37 ℃, the shaking table of 200r.p.m. rotating speed.Second day, the inoculum size with 2% was inoculated into new containing in 500 μ l/ hole LB substratum, the 96 hole micro plates of (adding 50 μ g/ml kalamycin resistances) (this is called daughter board below plate) with the bacterium liquid in each hole on the motherboard; Simultaneously, each hole adds 10% glycerine in the motherboard, and-80 ℃ of preservations are in order to use from now on.Daughter board is cultivated in 37 ℃, 220r.p.m. shaking table, treated that bacterium liquid reaches OD in each hole 600Be about about 0.5, the IPTG that adds 0.1mmol/L induces, induce 4h after, centrifugal 10 minutes of 4750r.p.m., thalline is collected in washing.The daughter board after centrifugal is put into-80 ℃ spend the night after, each hole adds phosphoric acid salt lysis buffer (PBS (pH7.4) 0.1mol/L, the MgCl of 250 μ l 210mmol/L, lysozyme 0.5mg/ml, DNaseI 2U/ml), be put in 37 ℃ and cultivate 40-60min.At this moment, because cold and hot expansion, the somatic cells wall is broken, and its zymoprotein that contains will be released in the liquid.4750r.p.m. centrifugal 10 minutes, and with PBS (NaCl 8g/L, KCl 0.2g/L, Na 2HPO 412H 2O 3.63g/L, KH 2PO 40.24g/L pH 7.4) behind the solution washing, enzyme liquid is shifted, preserve stereoselective high flux screening after being used for.
2, stereoselective high flux screening
In order to screen the chiral selectivity of sudden change back positive colony, select for use (R)-with (S)-methyl mandelate is as substrate, on 96 hole Sptting plates, react.(R)-with (S)-methyl mandelate is added on respectively in the two corresponding row reaction micropores.Fully behind the mixing, at room temperature react.Because methyl mandelate can form acid ion under the hydrolysis of enzyme, the pH value of solution is descended, make bromine Thymus vulgaris Finland indicator become yellow, thereby the absorbancy under the 630nm is changed from initial blue-greenish colour.Therefore, the variation that directly records the absorbancy in each reacting hole under the 630nm with ELIASA just can be carried out primary dcreening operation to the chiral selectivity of each two mutants.
The reaction system of high flux screening:
Bromine Thymus vulgaris Finland concentration of indicator: 0.1-5mmol/L;
Crude enzyme liquid: 20-80 μ g;
(R)-methyl mandelate (being dissolved in the trimethyl carbinol, acetonitrile, Virahol or acetone): 10-50mmol/L;
(S)-methyl mandelate (being dissolved in the trimethyl carbinol, acetonitrile, Virahol or acetone): 10-50mmol/L;
Supply system to 250 μ l with PBS damping fluid (pH7.0-7.6).
Behind the reaction certain hour, the absorbancy under the observation 630nm changes, tentatively relatively the stereoselectivity of each two mutants.
First round high flux screening has obtained two mutants A3G4, A1H1, A2E3, and A3F12, A3A8, A4G3, A4F6, A6D3, A6A5, A7D11, A8A5 tentatively shows stereoselective raising;
Second takes turns high flux screening, has obtained two mutants B2G1, B2E3, and B2E10, B3E11, B3D12, B4B7, B4C9, B7F11, B8E6, B8G1, B9E11 tentatively shows stereoselective raising;
The third round high flux screening has obtained two mutants C1D2, C1F9, and C2G10, C3D6, C3C11, C6H10, C7G12, C7G1, C8B2, C8G1, C9B1 tentatively shows stereoselective raising;
The four-wheel high flux screening has obtained two mutants D1B5, D3D4, and D3E1, D3E11, D4A11, D4E5, D4G12, D4F9, D5D10, D5E1, D6G7, D6G6, D6F6 tentatively shows stereoselective raising.
3, stereoselective liquid phase is measured
Demonstrate the positive colony of clear superiority for the primary dcreening operation result, carry out shake-flask culture: the two mutants that picking is corresponding from the motherboard of preserving is in the test tube of 5ml LB substratum (kantlex solution 50 μ g/ml), and 37 ℃ are carried out overnight cultures.Second day, the inoculum size with 2% was inoculated into the 250ml that contains 50ml LB substratum with the bacterium liquid in the test tube and shakes (kantlex 50 μ g/ml) in the bottle, carried out under 37 ℃, 200r.p.m cultivates.Treat bacterium liquid OD 600When value reached 0.5-1.0, the IPTG solution that adds 0.1mmol/L carried out abduction delivering.Behind the 4h, centrifugal collection thalline, and wash with PBS (pH7.4) solution, thalline is collected in centrifugal back, is dissolved in 50mmol/L Tris-HCl (pH 8.0) solution of 3-8ml, carries out ultrasonication.Carry out after the fragmentation that centrifugal (4750r.p.m., 20min), enzyme liquid is used for hydrolysis reaction.
Reaction system:
(R)-and (S)-methyl mandelate raceme (being dissolved in the trimethyl carbinol, acetonitrile, Virahol or acetone):
10-100mmol/L；
Enzyme liquid: 20-100 μ l;
Complement to 2-5ml with Tris-HCl (pH=8.0);
In 20-50 ℃, reaction 2-12h, sampling detects.
Use performance liquid chromatography (HPLC) detect (SPD-20A, Shimadzu).The chromatographic column model is that (Daicel, the chiral column of 250mm * 4.6mm) detect with ultraviolet/variable-wavelenght detector AD-H; Detect wavelength 230nm; Moving phase is Virahol and normal hexane (containing millesimal trifluoroacetic acid), and the ratio of the two is 10: 90, and flow velocity is 1ml/min.
Through multiple sieve, obtained two mutants A6A5, B7F11, C8G1 and D3E11, its stereoselectivity from initial E=3.1, is brought up to E=5.3 respectively, E=8.8, E=14.0 and E=30.8, the enzymatic enantio-selectivity size of E value representative.
4, active liquid phase is measured
For the two mutants A6A5 that improves by the stereoselectivity that embodiment obtained, B7F11, C8G1 and D3E11 carry out active mensuration.
Reaction system:
(R)-and (S)-methyl mandelate raceme (being dissolved in the trimethyl carbinol, acetonitrile, Virahol or acetone):
10-100mmol/L；
Enzyme liquid: 20-100 μ l;
Complement to 2-5ml with Tris-HCl (pH=8.0);
In 20-50 ℃, reaction 1h, sampling detects.
Use performance liquid chromatography (HPLC) detect (SPD-20A, Shimadzu).The chromatographic column model is that (Daicel, the chiral column of 250mm * 4.6mm) detect with ultraviolet/variable-wavelenght detector AD-H; Detect wavelength 230nm; Moving phase is Virahol and normal hexane (containing millesimal trifluoroacetic acid), and the ratio of the two is 10: 90, and flow velocity is 1ml/min.
It is a unit (1U) that the definition PM consumes the needed enzyme amount of 1 μ mol substrate.
Its activity from initial 10U/mg, is brought up to 43U/mg respectively, 142U/mg, 88U/mg, 20U/mg.
The sequential analysis of embodiment 3 esterase two mutants
To check order by two mutants A6A5, B7F11, C8G1 and the D3E11 that embodiment 2 is obtained (accomplishing) by Shanghai Ying Jin company; And use clustalxl 1.8 softwares and vector software to carry out the sequence comparison and analysis, analyze and obtain the esterase mutant genes shown in SEQ NO.1 in the sequence table, SEQ NO.2, SEQ NO.3 and the SEQ NO.4.
The ideal two mutants is carried out gene order and aminoacid sequence comparison, obtains the result and see table 1:
The The sequencing results of table 1 two mutants
Figure BSA00000141307700081
Sequence table
< 110>Zhejiang University
< 120>RSP_2728 esterase mutant genes that obtains through the orthogenesis means and esterase are at methyl mandelate
Application in the resolution reaction
<160>11
<210>1
<211>663
<212>DNA
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes that obtains through the orthogenesis means
<400>1
atgacacgca agctgacgtt cggccgccga ggcgcggccc cgggcgaggc cacgagcctc 60
gtcgtcttcc tccacggcta cggcgcggac ggggcggatc tcttgggtct ggccgagccg 120
cttgcgccgc acctgcccgg cacggccttc gtggcgcccg acgcgcccga gccctgccgc 180
gcctacggct tcggcttcca gtggtttccg atcccctggc tcgacggctc ctcggagacc 240
gccgccgccg aaggcatggc cgccgccgcc cgggatctcg acgccttcct cgacgagcgg 300
ctggccgagg aggggctgcc gcccgaggcg ctggcgctcg tgggcttctc gcagggcacg 360
atgatggcgc ttcatgtggc gccgcgccgg gccgaggaga tcgcgggcat cgtgggcttc 420
tcgggccgtc tgctcgcgcc ggagcggctg gccgaagagg cgcggtcgaa gccgccggtg 480
ctcctcgtgc atggcgacgc cgacccggtg gtgcccttcg ccgacatgag ccttgcgggc 540
gaggcgctgg ccgaggcggg cttcaccacc tacggccatg tgatgaaggg caccggccac 600
ggcatcgcgc ccgacggtct ttcggtggcg ctggccttcc tgaaggagcg tctgccggac 660
tga 663
<210>2
<211>663
<212>DNA
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes that obtains through the orthogenesis means
<400>2
atgacacgca agctgacgtt cggccgccga ggcgcggccc cgggcgaggc cacgagcctc 60
gtcgtcttcc tccacggcta cggcgcggac ggggcggatc tcttgggtct ggccgagccg 120
cttgcgccgc acctgcccgg cacggccttc gtggcgcccg acgcgcccga gccctgccgc 180
gcctacggct tcggcttcca gtggtttccg atcccctggc tcgacggctc ctcggagacc 240
gccgccgccg aaggcatggc cgccgccgcc cgggatctcg acgccttcct cgacgagcgg 300
ctggccgagg aggggctgcc gcccgaggcg ctggcgctcg tgggcttctc gcagggcacg 360
atgatggcgc ttcatgtggc gccgcgccgg gccgaggaga tcgcgggcat cgtgggcttc 420
tcgggccgtc tgcacgcgcc ggagcggctg gccgaagagg cgcggtcgaa gccgccggtg 480
ctcctcgtgc atggcgacgc cgacccggtg gtgcccttcg ccgacatgag ccttgcgggc 540
gaggcgctgg ccgaggcggg cttcaccacc tacggccatg tgatgaaggg caccggccac 600
ggcatcgcgc ccgacggtct ttcggtggcg ctggccttcc tgaaggagcg tctgccggac 660
tga 663
<210>3
<211>663
<212>DNA
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes that obtains through the orthogenesis means
<400>3
atgacacgca agctgacgtt cggccgccga ggcgcggccc cgggcgaggc cacgagcctc 60
gtcgtcttcc tccacggcta cggcgcggac ggggcggatc tcttgggtct ggccgagccg 120
cttgcaccgc acctgcccgg cacggccttc gtggcgcccg acgcgccaga gccctgccgc 180
gcctgcggct tcggcttcca gtggtttccg atcccctggc tcgacggctc ctcggagacc 240
gccgccgccg aaggcatggc cgccgccgcc cgggatctcg acgccttcct cgacgagcgg 300
ctggccgagg aggggctgcc gcccgaggcg ctggcgctcg tgggcttctc gcagggcacg 360
atgatggcgc ttcatgtggc gccgcgccgg gccgaggaga tcgcgggcat cgtgggcttc 420
tcgggccgtc tgcatgcgcc ggagcggctg gccgaagagg cgcggtcgaa gccgccggtg 480
ctcctcgtgc atggcgacgc cgacccggtg gtgcccttcg ccgacatgag ccttgcgggc 540
gaggcgctgg ccgaggcggg cttcaccacc tacggccatg tgatgaaggg caccggccac 600
ggcatcgcgc ccgacggtct ttcggtggcg ctggccttcc tgaaggagcg tctgccggac 660
tga 663
<210>4
<211>663
<212>DNA
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes that obtains through the orthogenesis means
<400>4
atgacacgca agctgacgtt cggccgccga ggcgcggccc cgggcgaggc cacgagcctc 60
gtcgtcttcc tccacggcta cggcgcggac ggggcggatc tcttgggtct ggccgagccg 120
cttgcaccgc acctgcccgg cacggccttc gtggcgcccg acgcgccaga gccctgccgc 180
gcctgcggct tcggcttcca gtggtttccg atcccctggc tcgacggctc ctcggagacc 240
gccgccgccg aaggcatggc cgccgccgcc cgggatctcg acgccttcct cgacgagcgg 300
ctggccgagg aggggctgcc gcccgaggcg ctggcgctcg tgggcttctc gcagggcacg 360
gtgatggcgc ttcatgtggc gccgcgccgg gccgaggaga tcgcgggcat cgtgggcttc 420
tcgggccgtc tgcatgcgcc ggagcggctg gccgaagagg cgcggtcgaa gccgccggtg 480
ctcctcgtgc atggcgacgc cgacccggtg gtgcccttcg ccgacatgag ccttgcgggc 540
gaggcgctgg ccgaggcggg cttcaccacc tacggccatg tgatgaaggg caccggccac 600
ggcatcgcgc ccgacggtct ttcg，ggcg ctggccttcc tgaaggagcg cctgccggac 660
tga 663
<210>5
<211>220
<212>PRT
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes amino acid sequence coded that obtains through the orthogenesis means
<400>5
Met Thr Arg Lys Leu Thr Phe Gly Arg Arg Gly Ala Ala Pro Gly Glu
1 5 10 15
Ala Thr Ser Leu Val Val Phe Leu His Gly Tyr Gly Ala Asp Gly Ala
20 25 30
Asp Leu Leu Gly Leu Ala Glu Pro Leu Ala Pro His Leu Pro Gly Thr
35 40 45
Ala Phe Val Ala Pro Asp Ala Pro Glu Pro Cys Arg Ala Tyr Gly Phe
50 55 60
Gly Phe Gln Trp Phe Pro Ile Pro Trp Leu Asp Gly Ser Ser Glu Thr
65 70 75 80
Ala Ala Ala Glu Gly Met Ala Ala Ala Ala Arg Asp Leu Asp Ala Phe
85 90 95
Leu Asp Glu Arg Leu Ala Glu Glu Gly Leu Pro Pro Glu Ala Leu Ala
100 105 110
Leu Val Gly Phe Ser Gln Gly Thr Met Met Ala Leu His Val Ala Pro
115 120 125
Arg Arg Ala Glu Glu Ile Ala Gly Ile Val Gly Phe Ser Gly Arg Leu
130 135 140
Leu Ala Pro Glu Arg Leu Ala Glu Glu Ala Arg Ser Lys Pro Pro Val
145 150 155 160
Leu Leu Val His Gly Asp Ala Asp Pro Val Val Pro Phe Ala Asp Met
165 170 175
Ser Leu Ala Gly Glu Ala Leu Ala Glu Ala Gly Phe Thr Thr Tyr Gly
180 185 190
His Val Met Lys Gly Thr Gly His Gly Ile Ala Pro Asp Gly Leu Ser
195 200 205
Val Ala Leu Ala Phe Leu Lys Glu Arg Leu Pro Asp
210 215 220
<210>6
<211>220
<212>PRT
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes amino acid sequence coded that obtains through the orthogenesis means
<400>6
Met Thr Arg Lys Leu Thr Phe Gly Arg Arg Gly Ala Ala Pro Gly Glu
1 5 10 15
Ala Thr Ser Leu Val Val Phe Leu His Gly Tyr Gly Ala Asp Gly Ala
20 25 30
Asp Leu Leu Gly Leu Ala Glu Pro Leu Ala Pro His Leu Pro Gly Thr
35 40 45
Ala Phe Val Ala Pro Asp Ala Pro Glu Pro Cys Arg Ala Tyr Gly Phe
50 55 60
Gly Phe Gln Trp Phe Pro Ile Pro Trp Leu Asp Gly Ser Ser Glu Thr
65 70 75 80
Ala Ala Ala Glu Gly Met Ala Ala Ala Ala Arg Asp Leu Asp Ala Phe
85 90 95
Leu Asp Glu Arg Leu Ala Glu Glu Gly Leu Pro Pro Glu Ala Leu Ala
100 105 110
Leu Val Gly Phe Ser Gln Gly Thr Met Met Ala Leu His Val Ala Pro
115 120 125
Arg Arg Ala Glu Glu Ile Ala Gly Ile Val Gly Phe Ser Gly Arg Leu
130 135 140
His Ala Pro Glu Arg Leu Ala Glu Glu Ala Arg Ser Lys Pro Pro Val
145 150 155 160
Leu Leu Val His Gly Asp Ala Asp Pro Val Val Pro Phe Ala Asp Met
165 170 175
Ser Leu Ala Gly Glu Ala Leu Ala Glu Ala Gly Phe Thr Thr Tyr Gly
180 185 190
His Val Met Lys Gly Thr Gly His Gly Ile Ala Pro Asp Gly Leu Ser
195 200 205
Val Ala Leu Ala Phe Leu Lys Glu Arg Leu Pro Asp
210 215 220
<210>7
<211>220
<212>PRT
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes amino acid sequence coded that obtains through the orthogenesis means
<400>7
Met Thr Arg Lys Leu Thr Phe Gly Arg Arg Gly Ala Ala Pro Gly Glu
1 5 10 15
Ala Thr Ser Leu Val Val Phe Leu His Gly Tyr Gly Ala Asp Gly Ala
20 25 30
Asp Leu Leu Gly Leu Ala Glu Pro Leu Ala Pro His Leu Pro Gly Thr
35 40 45
Ala Phe Val Ala Pro Asp Ala Pro Glu Pro Cys Arg Ala Cys Gly Phe
50 55 60
Gly Phe Gln Trp Phe Pro Ile Pro Trp Leu Asp Gly Ser Ser Glu Thr
65 70 75 80
Ala Ala Ala Glu Gly Met Ala Ala Ala Ala Arg Asp Leu Asp Ala Phe
85 90 95
Leu Asp Glu Arg Leu Ala Glu Glu Gly Leu Pro Pro Glu Ala Leu Ala
100 105 110
Leu Val Gly Phe Ser Gln Gly Thr Met Met Ala Leu His Val Ala Pro
115 120 125
Arg Arg Ala Glu Glu Ile Ala Gly Ile Val Gly Phe Ser Gly Arg Leu
130 135 140
His Ala Pro Glu Arg Leu Ala Glu Glu Ala Arg Ser Lys Pro Pro Val
145 150 155 160
Leu Leu Val His Gly Asp Ala Asp Pro Val Val Pro Phe Ala Asp Met
165 170 175
Ser Leu Ala Gly Glu Ala Leu Ala Glu Ala Gly Phe Thr Thr Tyr Gly
180 185 190
His Val Met Lys Gly Thr Gly His Gly Ile Ala Pro Asp Gly Leu Ser
195 200 205
Val Ala Leu Ala Phe Leu Lys Glu Arg Leu Pro Asp
210 215 220
<210>8
<211>220
<212>PRT
< 213>artificial sequence
<220>
< 223>the RSP_2728 esterase mutant genes amino acid sequence coded that obtains through the orthogenesis means
<400>8
Met Thr Arg Lys Leu Thr Phe Gly Arg Arg Gly Ala Ala Pro Gly Glu
1 5 10 15
Ala Thr Ser Leu Val Val Phe Leu His Gly Tyr Gly Ala Asp Gly Ala
20 25 30
Asp Leu Leu Gly Leu Ala Glu Pro Leu Ala Pro His Leu Pro Gly Thr
35 40 45
Ala Phe Val Ala Pro Asp Ala Pro Glu Pro Cys Arg Ala Cys Gly Phe
50 55 60
Gly Phe Gln Trp Phe Pro Ile Pro Trp Leu Asp Gly Ser Ser Glu Thr
65 70 75 80
Ala Ala Ala Glu Gly Met Ala Ala Ala Ala Arg Asp Leu Asp Ala Phe
85 90 95
Leu Asp Glu Arg Leu Ala Glu Glu Gly Leu Pro Pro Glu Ala Leu Ala
100 105 110
Leu Val Gly Phe Ser Gln Gly Thr Val Met Ala Leu His Val Ala Pro
115 120 125
Arg Arg Ala Glu Glu Ile Ala Gly Ile Val Gly Phe Ser Gly Arg Leu
130 135 140
His Ala Pro Glu Arg Leu Ala Glu Glu Ala Arg Ser Lys Pro Pro Val
145 150 155 160
Leu Leu Val His Gly Asp Ala Asp Pro Val Val Pro Phe Ala Asp Met
165 170 175
Ser Leu Ala Gly Glu Ala Leu Ala Glu Ala Gly Phe Thr Thr Tyr Gly
180 185 190
His Val Met Lys Gly Thr Gly His Gly Ile Ala Pro Asp Gly Leu Ser
195 200 205
Val Ala Leu Ala Phe Leu Lys Glu Arg Leu Pro Asp
210 215 220
<210>9
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>fallibility pcr amplification primer S2a
<400>9
cgggatccat gacacgcaag ctgacgttcg 30
<210>10
<211>31
<212>DNA
< 213>artificial sequence
<220>
< 223>fallibility pcr amplification primer S2b
<400>10
cccaagcttt cagtccggca gacgctcctt c 31
<210>11
<211>663
<212>DNA
< 213>type red bacterium of ball (Rhodobacter sphaeroides)
<220>
< 223>the RSP_2728 wild type gene of type red bacterium Rhodobacter of ball sphaeroides
<400>11
atgacacgca agctgacgtt cggccgccga ggcgcggccc cgggcgaggc cacgagcctc 60
gtcgtcttcc tccacggcta cggcgcggac ggggcggatc tcttgggtct ggccgagccg 120
cttgcgccgc acctgcccgg cacggccttc gtggcgcccg acgcgcccga gccctgccgc 180
gccaacggct tcggcttcca gtggtttccg atcccctggc tcgacggctc ctcggagacc 240
gccgccgccg aaggcatggc cgccgccgcc cgggatctcg acgccttcct cgacgagcgg 300
ctggccgagg aggggctgcc gcccgaggcg ctggcgctcg tgggcttctc gcagggcacg 360
atgatggcgc ttcatgtggc gccgcgccgg gccgaggaga tcgcgggcat cgtgggcttc 420
tcgggccgtc tgctcgcgcc ggagcggctg gccgaagagg cgcggtcgaa gccgccggtg 480
ctcctcgtgc atggcgacgc cgacccggtg gtgcccttcg ccgacatgag ccttgcgggc 540
gaggcgctgg ccgaggcggg cttcaccacc tacggccatg tgatgaaggg caccggccac 600
ggcatcgcgc ccgacggtct ttcggtggcg ctggccttcc tgaaggagcg tctgccggac 660
tga 663

Claims (10)

1. the mutator gene of RSP 2728 genes of the red bacterium of a kind ball (Rhodobacter sphaeroides) is characterized in that the nucleotide sequence of said mutator gene is one of following nucleotide sequences:
(1) nucleotide sequence of SEQ ID NO:1 in the sequence table;
(2) nucleotide sequence of SEQ ID NO:2 in the sequence table;
(3) nucleotide sequence of SEQ ID NO:3 in the sequence table;
(4) nucleotide sequence of SEQ ID NO:4 in the sequence table.
2. esterase is characterized in that: said esterase is by mutator gene coding in the claim 1, and the aminoacid sequence of said esterase is one of following amino acid sequences:
(1) aminoacid sequence of SEQ ID NO:5 in the sequence table;
(2) aminoacid sequence of SEQ ID NO:6 in the sequence table;
(3) aminoacid sequence of SEQ ID NO:7 in the sequence table;
(4) aminoacid sequence of SEQ ID NO:8 in the sequence table;
Wherein, the esterase called after XLA in the sequence table shown in the aminoacid sequence of SEQ ID NO:5 becomes tyrosine with respect to its asparagine mutation of the 62nd of wild-type; Esterase called after XLB in the sequence table shown in the aminoacid sequence of SEQ ID NO:6 becomes tyrosine, the 145th leucine to be mutated into Histidine with respect to its asparagine mutation of the 62nd of wild-type; Esterase called after XLC in the sequence table shown in the aminoacid sequence of SEQ ID NO:7 becomes halfcystine, the 145th leucine to be mutated into Histidine with respect to its 62nd asparagine mutation of wild-type; Esterase called after XLD in the sequence table shown in the aminoacid sequence of SEQ ID NO:8 becomes with respect to its 62nd asparagine mutation of wild-type that halfcystine, the 121st methionine(Met) are mutated into a word used in person's names propylhomoserin, the 145th leucine is mutated into Histidine.
3. the preparation method of esterase XLA, XLB, XLC or XLD in the claim 2 is characterized in that:
According to the nucleotide sequence in the claim 1 design primer, utilize the method for pcr amplification to obtain amplified production and with it with after expression vector pET-30a (+) links to each other, be transformed in the e. coli bl21 host cell the above-mentioned esterase of acquisition behind abduction delivering.
4. the application of the described esterase of claim 2 is characterized in that: the resolution reaction that said esterase is used for methyl mandelate.
5. the application of esterase according to claim 4 is characterized in that: when for esterase XLA, from wild-type, enantio-selectivity has been brought up to E=5.3 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it; It from wild-type, has brought up to 43U/mg from 10U/mg to the activity of methyl mandelate.
6. the application of esterase according to claim 4 is characterized in that: when for esterase XLB, from wild-type, enantio-selectivity has been brought up to E=8.8 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it; It from wild-type, has brought up to 142U/mg from 10U/mg to the activity of methyl mandelate.
7. the application of esterase according to claim 4; It is characterized in that: when the time for esterase XLC; Its to (S)-type methyl mandelate stereoselectivity from wild-type; Enantio-selectivity has been brought up to E=14.0 from E=3.1, and it from wild-type, has brought up to 88U/mg from 10U/mg to the activity of methyl mandelate.
8. the application of esterase according to claim 4 is characterized in that: when for esterase XLD, from wild-type, enantio-selectivity has been brought up to E=30.8 from E=3.1 to (S)-type methyl mandelate stereoselectivity for it; It from wild-type, has brought up to 20U/mg from 10U/mg to the activity of methyl mandelate.
9. a carrier is characterized in that comprising the described mutator gene of claim 1.
10. a host cell is characterized in that comprising the described carrier of claim 9.
CN2010101868653A 2010-05-28 2010-05-28 RSP_2728 esterase mutant genes obtained by directed evolution and application of esterase in methyl mandelate resolution reaction Expired - Fee Related CN101870984B (en)

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