CN101870984A - 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 PDFInfo
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- CN101870984A CN101870984A CN201010186865A CN201010186865A CN101870984A CN 101870984 A CN101870984 A CN 101870984A CN 201010186865 A CN201010186865 A CN 201010186865A CN 201010186865 A CN201010186865 A CN 201010186865A CN 101870984 A CN101870984 A CN 101870984A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention belongs to the fields of gene engineering and enzyme engineering, and discloses mutant genes of an esterase gene RSP-2728 and an application thereof in the aspect of gene engineering. The ultimate mutant esterases XLA, XLB, XLC and XLD are obtained after four rounds of screening by directed evolution, an error-prone PCR method, a gene cloning method and a high-throughput screening method applicable to activity and stereoselectivity. The application of the mutant esterases in the resolution reaction of the methyl mandelate confirms that compared with a wild type, the mutant esterase XLD of which the stereoselectivity is the highest has the enantiomer selectivity (E value) increased from 3.1 to 30.8 in the aspect of stereoselectivity; and compared with a wild type, the activity of the XLB which has the highest reaction activity to the substrate is increased from 10U/mg to 142U/mg. The invention has better application prospects in the process of substrate racemization or synthesis of important medicine intermediates.
Description
Technical field
The invention belongs to enzyme engineering and genetically engineered field, relate in particular to the mutant that obtains to come from the esterase RSP_2728 gene (GeneID:3720458) of the red bacterium of class ball (Rhodobacter sphaeroides) bacterial strain by 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 the crucial biological catalyst of a class, 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 by correlation technique has great importance.Under the situation to enzymatic structure or catalytic mechanism poor information, the orthogenesis technology provides strong instrument for developing new biocatalysis.Many studies show that adopts 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 by the orthogenesis means.
The present invention also aims to provide a kind of esterase and application thereof of encoding by 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 described 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 described 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 described esterase is used for methyl mandelate, when being esterase XLA, its to (S)-type methyl mandelate stereoselectivity from wild-type, enantio-selectivity has been brought up to E=5.3 from E=3.1, 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 will contain esterase gene imports to the host; Described host is a bacillus coli DH 5 alpha, one of BL21 or JM109.
A kind of is indicator with bromine Thymus vulgaris Finland, determines the high-throughput screening method that enzyme is lived by the monitoring hydrogen ion concentration that ester hydrolysis reaction produced.
The RSP_2728 wild type gene of the red bacterium of class 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 by 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 by 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, reclaim test kit with the PCR purifying and carry out the purifying recovery 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, select to have and insert segmental clone, 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 mutant
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 plate is hereinafter referred to as motherboard), with motherboard overnight incubation 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 plate is hereinafter referred to as daughter board) 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 using 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, since cold and hot expansion, the fragmentation of somatic cells wall, 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)-and (S)-methyl mandelate is as substrate, on 96 hole Sptting plates, reacts.(R)-and (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, directly the variation that records the absorbancy in each reacting hole under the 630nm with microplate reader just can be carried out primary dcreening operation to the chiral selectivity of each mutant.
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 mutant.
First round high flux screening has obtained mutant A3G4, A1H1, A2E3, and A3F12, A3A8, A4G3, A4F6, A6D3, A6A5, A7D11, A8A5 tentatively shows stereoselective raising;
Second takes turns high flux screening, has obtained mutant B2G1, B2E3, and B2E10, B3E11, B3D12, B4B7, B4C9, B7F11, B8E6, B8G1, B9E11 tentatively shows stereoselective raising;
The third round high flux screening has obtained mutant C1D2, C1F9, and C2G10, C3D6, C3C11, C6H10, C7G12, C7G1, C8B2, C8G1, C9B1 tentatively shows stereoselective raising;
The four-wheel high flux screening has obtained mutant D1B5, D3D4, and D3E1, D3E11, D4A11, D4E5, D4G12, D4F9, D5D10, D5E1, D6G7, D6G6, D6F6 tentatively shows stereoselective raising.
3, stereoselective liquid phase is measured
Present the positive colony of clear superiority for the primary dcreening operation result, carry out shake-flask culture: the mutant of picking correspondence is in the test tube of 5ml LB substratum (kantlex solution 50 μ g/ml) from the motherboard of preserving, and 37 ℃ are carried out overnight incubation.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 high 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.
By multiple sieve, obtained mutant 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 mutant 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 high 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 per minute 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 mutant
To check order by mutant A6A5, B7F11, C8G1 and the D3E11 that embodiment 2 is obtained (finishing) 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 mutant is carried out gene order and aminoacid sequence comparison, obtains to the results are shown in Table 1:
The The sequencing results of table 1 mutant
Sequence table
＜110〉Zhejiang University
＜120〉RSP_2728 esterase mutant genes that obtains by 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 by 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 by 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 by 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 by 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 by 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 by 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 by 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 by 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〉the red bacterium of class ball (Rhodobacter sphaeroides)
<220>
＜223〉the RSP_2728 wild type gene of the red bacterium Rhodobacter of class 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 Rhodobacter of kind ball sphaeroides is characterized in that,
The nucleotide sequence of described 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: described esterase is by mutator gene coding in the claim 1, and the aminoacid sequence of described 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.
3. the preparation method of esterase XLA, XLB, XLC or XLD in the claim 2, it is characterized in that: according to the design of the nucleotide sequence in the claim 1 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.
4. the application of the described esterase of claim 2 is characterized in that: the resolution reaction that described 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, 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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820521A (en) * | 2013-11-27 | 2014-05-28 | 浙江大学 | Method for preparing R-o-chloromandelic acid methyl ester through biocatalysis dynamic kinetic resolution |
| CN105349507A (en) * | 2015-12-15 | 2016-02-24 | 中国科学院南海海洋研究所 | Lipase LIPDa6 as well as encoding gene and application thereof |
| CN113201516A (en) * | 2021-04-28 | 2021-08-03 | 安徽丰乐香料有限责任公司 | P-nitrobenzyl esterase mutant and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509028A (en) * | 2009-03-23 | 2009-08-19 | 东华大学 | Method for orthogenesis with differ-degree continuous-fallibility PCR |
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| CN101509028A (en) * | 2009-03-23 | 2009-08-19 | 东华大学 | Method for orthogenesis with differ-degree continuous-fallibility PCR |
Non-Patent Citations (2)
| Title |
|---|
| 《Current Opinion in Biotechnology》 20021031 Uwe T Bornscheuer Methods to increase enantioselectivity of lipases and esterases 543-547 1-10 , 2 * |
| 《Current Opinion in Biotechnology》 20040717 Karl-Erich Jaeger et al. Enantioselectivity biocatalysis optimized by directed evolution 305-313 1-10 , 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820521A (en) * | 2013-11-27 | 2014-05-28 | 浙江大学 | Method for preparing R-o-chloromandelic acid methyl ester through biocatalysis dynamic kinetic resolution |
| CN105349507A (en) * | 2015-12-15 | 2016-02-24 | 中国科学院南海海洋研究所 | Lipase LIPDa6 as well as encoding gene and application thereof |
| CN105349507B (en) * | 2015-12-15 | 2018-09-28 | 中国科学院南海海洋研究所 | A kind of lipase LIPDa6 and its encoding gene and application |
| CN113201516A (en) * | 2021-04-28 | 2021-08-03 | 安徽丰乐香料有限责任公司 | P-nitrobenzyl esterase mutant and application thereof |
| CN113201516B (en) * | 2021-04-28 | 2022-05-27 | 安徽丰乐香料有限责任公司 | P-nitrobenzyl esterase mutant and application thereof |
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| CN101870984B (en) | 2012-06-13 |
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