CN102796719B - (+)Gamma-lactamase, its coding gene and application - Google Patents
(+)Gamma-lactamase, its coding gene and application Download PDFInfo
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- CN102796719B CN102796719B CN201210293158.3A CN201210293158A CN102796719B CN 102796719 B CN102796719 B CN 102796719B CN 201210293158 A CN201210293158 A CN 201210293158A CN 102796719 B CN102796719 B CN 102796719B
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Abstract
The invention discloses an Aeropyrum pernix derived (+)gamma-lactamase, its coding gene and application. The (+)gamma-lactamase provided in the invention is the protein of following (a) or (b): (a) a protein composed of an amino acid residue sequence in SEQ ID No:1 of a sequence table; and (b) an SEQ ID No:1 derived protein that is composed of an amino acid residue sequence obtained by subjecting the SEQ ID No:1 amino acid residue sequence in the sequence table to substitution and/or deletion and/or adding by one or multiple amino acid residues and has racemate gamma-lactam resolution activity. By applying the (+)gamma-lactamase to hydrolytic resolution of the racemate gamma-lactam, (-)gamma-lactam with optical purity of 91.4%-99.9% can be obtained, and the yield is more than 45%.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of (+) gamma-lactam enzyme and encoding gene and application with resolving racemic gamma-lactam activity.
Background technology
(-) 2-azabicyclo [2,2,1] heptan-5-alkene-3-ketone (being called for short (-) gamma-lactam) is that synthetic antiviral is as the important intermediate of Abacavir, anti-influenza A and bird flu medicine Peramivir.Be method preparation (-) gamma-lactam by chemosynthesis traditionally, but the method cost is high, complex steps, and the heavy metal catalyst using in catalytic process can cause serious pollution to environment.And biological enzyme has the plurality of advantages such as high-efficient simple, save energy, environmental friendliness in the process of synthesis of chiral (-) gamma-lactam.
Enzyme that can Hydrolysis Resolution raceme gamma-lactam is called as (+) gamma-lactam enzyme.(+) gamma-lactam enzyme belongs to a kind of of Ntn hydrolase, gamma-lactam enzyme be mainly used in the chirality of (-) gamma-lactam synthetic in.(+) gamma-lactam enzyme of report mainly contains the shortcoming existing in the split process of racemization gamma-lactam at present: the selectivity that (1) (+) gamma-lactam enzyme splits racemization gamma-lactam is poor; (2) enzyme of selecting is unstable, thereby has limited its industrial applications.
Summary of the invention
The object of this invention is to provide a kind of (+) gamma-lactam enzyme and encoding gene and application with resolving racemic gamma-lactam activity.Should to (+) gamma-lactam, there is absolutely selective by (+) gamma-lactam enzyme, selective hydrolysis (+) gamma-lactam during catalyzed reaction, thus obtain optically pure (-) gamma-lactam, synthetic for medicine.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
(+) provided by the invention gamma-lactam enzyme is named as A-P, derives from raw archeobacteria (Aeropyrum pernix) the NBRC No.100138 of thermophilic spring.
(+) gamma-lactam enzyme A-P is following (a) or protein (b):
(a) a kind of protein of the amino acid residue sequence that comprises SEQ ID No:1;
(b) amino acid residue sequence that comprises SEQ ID No:1 is through replacing and/or disappearance and/or add at least one or more amino-acid residue and the amino acid residue sequence that obtains, and has the protein of resolving racemic gamma-lactam activity.
Wherein, the SEQ ID No:1 in sequence table is comprised of 377 amino-acid residues.
The encoding gene of above-mentioned (+) gamma-lactam enzyme also belongs to protection scope of the present invention, and it can have one of following nucleotide sequence:
(a) nucleotide sequence shown in SEQ ID No:2 in sequence table;
(b) polynucleotide of SEQ ID No:1 protein sequence in code sequence list.
Wherein, the SEQ ID No:2 in sequence table is by 1134 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID No:1 in sequence table from 5 ' end the 1st to the 1134th bit base.
The object of the invention is to realize by following another technical scheme:
The expression vector that contains gene of the present invention, clone and Host Strains all belong to protection scope of the present invention.
The preparation method of (+) of the present invention gamma-lactam enzyme A-P is:
A. with raw archeobacteria (Aeropyrum pernix) the NBRC No.100138 of thermophilic spring genome, design primer for masterplate, carry out polymerase chain reaction (PCR) amplification gene, amplified fragments obtained above is carried out to 1.5% agarose gel electrophoresis, after electrophoresis, reclaim the band that size is about 1200bp, obtain object fragment;
B. above-mentioned purpose fragment is connected on T carrier with T4 ligase enzyme, be transformed in intestinal bacteria E.coli DH5 α, converted product is coated onto on blue hickie flat board, wherein in substratum, add the bromo-4 chloro-3-indoles-β-D-galactosides (X-gal) of 40 μ L 5-, 8 μ L 500mol/L sec.-propyl-β-D-sulfo-galactopyranosides (IPTG), 37 ℃ of incubated overnight;
C. the single bacterium colony after the above-mentioned incubated overnight of picking carries out PCR, according to electrophoresis result, select single bacterium colony of order-checking, to check order successful single bacterium colony in 37 ℃ of cultivations, and carry out carrying in plasmid, with Nde I and Hind III restriction endonuclease, object fragment and plasmid pET30 being carried out to enzyme cuts, carry out 1.5% agarose gel electrophoresis, after electrophoresis, reclaim respectively object fragment and carrier large fragment.
D. above-mentioned object fragment is connected in carrier large fragment with T4 ligase enzyme, is again transformed in E.coli DH5 α, converted product is coated onto on the flat board containing kantlex, 37 ℃ of incubated overnight.
E. the single bacterium colony after the above-mentioned incubated overnight of picking carries out PCR, according to electrophoresis result, selects order-checking band, and the successful single bacterium colony that will check order is in 37 ℃ of cultivations, and carries out carrying in plasmid, obtains the expression vector building.
F. the above-mentioned expression vector building is imported to host cell, with 60 μ L 500mol/L IPTG abduction deliverings, obtain (+) gamma-lactam enzyme A-P.
(+) of the present invention gamma-lactam enzyme A-P can also be by the encoding gene of chemosynthesis (+) gamma-lactam enzyme A-P, gene constructed to expression vector by conventional genetic manipulation means by this chemosynthesis, and import e. coli host cell and obtain transformant, cultivate this transformant, transformant after cultivation is recombination bacillus coli thalline, expresses and obtains (+) gamma-lactam enzyme A-P.
For building the recombinant expression vector that contains above-mentioned gamma-lactam enzyme coding gene, can be the plasmid expression vector in genetically engineered field, feasible carrier comprises pET serial carrier, pUC serial carrier, pGEX serial carrier etc.Host cell is selected and the corresponding host of above-mentioned expression vector, as intestinal bacteria E.coli BL-21 (DH3) etc.Albumen after expression can further utilize generally acknowledges that known purification process carries out purifying.
The object of the invention is to realize by following another technical scheme:
(+) of the present invention gamma-lactam enzyme A-P can be for the fractionation of the gamma-lactam of racemization, and its method is:
Recombination bacillus coli thalline or restructuring (+) gamma-lactam enzyme A-P are suspended in the phosphoric acid buffer of 150mmol pH6.0 ~ 8.5, to (-) gamma-lactam substrate that adds 5g/L in described phosphoric acid buffer, react 10 ~ 24 hours at 60 ~ 120 ℃.Above-mentioned reaction mixture is centrifugal, get supernatant liquor, to 250 μ L ethyl acetate in described supernatant liquor, extract, obtain described (-) gamma-lactam.
The present invention's beneficial effect is compared to existing technology:
1. the invention provides a kind of (+) gamma-lactam enzyme and encoding gene thereof with resolving racemic gamma-lactam activity, because this (+) gamma-lactam enzyme splits and has absolutely selective racemization gamma-lactam, so greatly improved purity and the productive rate of (-) gamma-lactam.
2. (+) provided by the invention gamma-lactam enzymatic property is stable, at 60 ~ 120 ℃, can keep for a long time its vigor, is compared to normal temperature enzyme and has the advantages that tolerance is strong, so possess the prospect of industrial applications.
3. (+) provided by the invention gamma-lactam enzyme has higher temperature tolerance, is compared to existing normal temperature enzyme, can take the method for high-temperature heat treatment, can a step obtain large-scale purification albumen, and method is simple, and cost is lower.
4. utilization of the present invention (+) gamma-lactam enzyme resolving racemic gamma-lactam, method high-efficient simple, environmental pollution is little.
Accompanying drawing explanation
Fig. 1. the DNA electrophorogram that the raw archeobacteria genome of the thermophilic spring that take is template pcr amplification A-P
Fig. 2. the SDS-PAGE figure that restructuring A-P expresses
Fig. 3. restructuring A-P resolution of racemic gamma-lactam HPLC collection of illustrative plates
Embodiment
(1) acquisition of (+) of the present invention gamma-lactam enzyme A-P gene
(1) (+) gamma-lactam enzyme A-P genome Japan Biological resources centers (NBRC) directly buys
(2) design of primers
According to known (+) gamma-lactam enzyme A-P gene order design primer, primer sequence following (synthetic by the raw work in Shanghai);
A-P upstream primer:
5’-GGAATTC
CATATGGTAACAAGGATAACT-3’
Underscore represents Nde I restriction enzyme site
A-P downstream primer:
5’-CCC
AAGCTTCTACTCTGCTAGCTTCTTC-3’
Underscore represents Hind III restriction enzyme site
(3) pcr amplification and gene clone
The described genomic dna of take carries out pcr amplification as template, PCR reaction system is: 1 μ L genomic dna (100 μ g/ μ L), 2.5 μ L10 * PCR Buffer, each 1 μ L of upstream and downstream primer, 1 μ L dNTP(2.5mM, GenStar, Cat#A114-01), 1mL Taq enzyme (5U/ μ L, TaKaRa Taq
tM, DR001A), use ddH
2o mends to 25 μ L.PCR condition is: 95 ℃ of the first step thermally denatures, 5 minutes; 95 ℃ of second step thermally denatures, 30 seconds; 53 ℃ of three-step annealings, 1 minute; The 4th step is extended 72 ℃, 1 minute; The 5th step is extended 72 ℃, 10 minutes.30 circulations are set between second step to the four steps, and after PCR reaction finishes, agarose gel electrophoresis detects and (sees accompanying drawing 1, swimming lane 1:A-P PCR product; Swimming lane M:DNA molecular weight standard).
PCR product reclaims test kit (QIAGEN, Cat.No.28704) through glue, and the specification sheets that working method provides according to test kit carries out, and reclaims the band of 1200bp left and right; After this band is reclaimed, with T4 ligase enzyme, be connected on T carrier, be transformed in E.coli DH5 α, converted product carries out blue hickie screening (adding 40 μ L X-gal in substratum, 8 μ L500mol/L IPTG), 37 ℃ of incubated overnight; The single bacterium colony obtaining after picking incubated overnight, carries out bacterium colony PCR, selects single bacterium colony of order-checking according to PCR product electrophoresis result, and the successful single bacterium colony that will check order is in 37 ℃ of cultivations, and carries out carrying in plasmid; The plasmid of extraction is used respectively to Nde I (Fermentas, Cat.No.FD0584) and Hind III enzyme cut (Fermentas, Cat.No.FD0504), be connected into the pET30 plasmid (Novagen that same enzyme is cut, Cat.No.69909-3), obtain the carrier pET30A-P that builds.
(2) expression of recombinant proteins and purifying
The described plasmid pET30A-P building is imported to intestinal bacteria E.coli BL-21(DH3 by heat shock method), obtain transformant E.coli BL-21(pET30A-P); Described transformant 800 μ L are inoculated in the test tube of LB liquid nutrient medium (containing kantlex, concentration is 30 μ g/mL) to 37 ℃ of incubated overnight; Then be inoculated in 50mL LB substratum, cultivate 3-4 hour for 37 ℃, add 60 μ L 500mol/L IPTG, 30 ℃ of abduction deliverings that spend the night, obtain abduction delivering mixed solution.Above-mentioned abduction delivering mixed solution is centrifugal and collect thalline, and this thalline is recombination bacillus coli thalline; Described thalline is suspended in phosphoric acid buffer to ultrasonication (227W, ultrasonic 2 seconds, 4.5 seconds, interval, ultrasonic 50 minutes).By the bacterium liquid of described ultrasonication centrifugal (12000rpm), collect supernatant liquor, obtain (+) gamma-lactam enzyme A-P crude extract.
According to the thermophilic characteristic of described (+) gamma-lactam enzyme A-P, purge process does not adopt the method for Ni-NTA affinity column purifying, but adopts the method for heat except foreigh protein removing.By described (+) gamma-lactam enzyme A-P crude extract obtaining after ultrasonication, in 80 ~ 100 ℃ of temperature ranges, heating is 10 ~ 24 hours, and the centrifugal precipitation of removing of 14800rpm obtains supernatant liquor, obtains described (+) gamma-lactam enzyme A-P.Through SDS-PAGE, detect and show that the purification effect of albumen is better, removed most foreign protein, detected result is shown in accompanying drawing 2(swimming lane M: molecular weight of albumen standard; Blank before swimming lane 1:pETA-P induction; After swimming lane 2:pETA-P induction, express; After swimming lane 3:pETA-P cytoclasis, supernatant is expressed; Swimming lane 4, pETA-P cytoclasis postprecipitation).
(3) application of (+) gamma-lactam enzyme in the gamma-lactam of racemization splits
The determination of activity of described (+) gamma-lactam enzyme adopts HPLC method.Chromatographic column model is the Chiralpark AS-H of Daicel company (250 * 4.6mm); Moving phase is acetonitrile: Virahol 9:1; Flow velocity is 0.6mL/min; Detection wavelength is 230nm.Measure the peak area of the lactan standard substance of different concns, drawing standard regression curve, thus can analyze according to peak area quantification content and the transformation efficiency of lactan.
Get (+) gamma-lactam enzyme A-P(described in 150 μ L approximately containing 10 μ g A-P) be suspended in the phosphoric acid buffer of 450 μ L 150mmolpH6.0 ~ 8.5, to (-) gamma-lactam substrate that adds 50 μ L 5g/L in described phosphoric acid buffer, react 10 ~ 24 hours at 60 ~ 120 ℃.Above-mentioned reaction mixture is centrifugal, get supernatant liquor, to 250 μ L ethyl acetate in described supernatant liquor, extract, obtain described (-) gamma-lactam.
Get described (-) gamma-lactam 100 μ L and carry out HPLC detection, obtaining optical purity is 91.4% ~ 99.9%, and yield is greater than 45%, and referring to Fig. 3, (HPLC of A raceme gamma-lactam analyzes collection of illustrative plates to result; B enzyme splits (-) gamma-lactam HPLC obtaining and analyzes collection of illustrative plates; Wherein, 1 ethyl acetate solvent peak, 2 (+) gamma-lactam, 3 (-) gamma-lactam).
Quality raw materials used in the present embodiment is identical with embodiment 1, preparation method is referring to embodiment 1, improvements are: (+) gamma-lactam enzyme splits in the gamma-lactam application of racemization, get in the phosphoric acid buffer that 5mg recombination bacillus coli thalline is suspended in 450 μ L 150mmol pH6.0 ~ 8.5, to (-) gamma-lactam substrate that adds 50 μ L 5g/L in described phosphoric acid buffer, react 10 ~ 24 hours at 60 ~ 120 ℃.Above-mentioned reaction mixture is centrifugal, get supernatant liquor, to 250 μ L ethyl acetate in described supernatant liquor, extract, obtain described (-) gamma-lactam.
Get described (-) gamma-lactam 100 μ L and carry out HPLC(Shimadzu, Essentia LC-15C) detect, obtaining optical purity is 91.4% ~ 99.9%, yield is greater than 45%.
The reagent adopting in embodiment 1, embodiment 2 and instrument are as being market conventional products as specified otherwise.
In embodiment 1, embodiment 2, the genetically engineered elementary operation methods such as PCR, the enzyme adopting cut, connected, conversion are recorded in the (work such as (U.S.) J. Pehanorm Brooker in < < molecular cloning experiment guide third edition > >, Science Press, publishes for 2002).
Obviously, the above embodiment of the present invention be only for clearly illustrate that the present invention does for example, and be not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation or changes.Here cannot give all embodiments exhaustive.Every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.
Claims (3)
1. a method of utilizing (+) gamma-lactam enzyme preparation (-) gamma-lactam, described (+) gamma-lactam enzyme is following protein (a):
(a) a kind of amino acid residue sequence is the protein of SEQ ID No:1;
Utilize the method for (+) gamma-lactam enzyme preparation (-) gamma-lactam to be:
Recombination bacillus coli thalline or restructuring (+) gamma-lactam enzyme A-P are suspended in phosphoric acid buffer, in described phosphoric acid buffer, add (-) gamma-lactam substrate, react 10～24 hours at 60～120 ℃, obtain reaction mixture; Above-mentioned reaction mixture is centrifugal, get supernatant liquor, in described supernatant liquor, add ethyl acetate to extract, obtain described (-) gamma-lactam;
Described (-) gamma-lactam is 2-azabicyclo [2,2,1] heptan-5-alkene-3-ketone.
2. utilize the method for (+) gamma-lactam enzyme coding gene resolving racemic gamma-lactam preparation (-) gamma-lactam, described (+) gamma-lactam enzyme coding gene is following nucleotide sequence (a) or (b):
(a) nucleotide sequence shown in SEQ ID No:2;
(b) polynucleotide sequence of coding SEQ ID No:1 protein sequence;
Utilize the method for (+) gamma-lactam enzyme coding gene resolving racemic gamma-lactam preparation (-) gamma-lactam to be:
With raw archeobacteria (Aeropyrum pernix) the NBRC No:100138 of thermophilic spring genome, design primer, upstream primer for masterplate: 5 '-GGAATTC
cATATGgTAACAAGGATAACT-3 '; Downstream primer: 5 '-CCC
aAGCTTcTACTCTGCTAGCTTCTTC-3 '; Carry out polymerase chain reaction (PCR) amplifying target genes, electrophoresis reclaims object fragment; Again described object fragment is connected on T carrier with T4 ligase enzyme, is transformed in E.coli DH5 α, successively dull and stereotyped by blue hickie, containing the dull and stereotyped screening of kantlex; Carry out afterwards single bacterium colony PCR, according to electrophoresis result, select single bacterium colony of order-checking, the 37 ℃ of cultivations of successful single bacterium colony of checking order, and carry out carrying in plasmid, obtain the expression vector building; Described expression vector is imported to host cell, with IPTG abduction delivering, obtain recombinant host bacterium thalline, described recombinant host bacterium thalline is carried out to broken (+) gamma-lactam enzyme A-P described in obtaining after centrifugal;
Described (+) gamma-lactam enzyme A-P is suspended in phosphoric acid buffer, in described phosphoric acid buffer, adds (-) gamma-lactam substrate, react 10～24 hours at 60～120 ℃.Above-mentioned reaction mixture is centrifugal, get supernatant liquor, in described supernatant liquor, add ethyl acetate to extract, obtain described (-) gamma-lactam;
Described (-) gamma-lactam is 2-azabicyclo [2,2,1] heptan-5-alkene-3-ketone.
3. utilize the method for (+) gamma-lactam enzyme coding gene resolving racemic gamma-lactam preparation (-) gamma-lactam, described (+) gamma-lactam enzyme coding gene is following nucleotide sequence (a) or (b):
(a) nucleotide sequence shown in SEQ ID No:2;
(b) polynucleotide sequence of coding SEQ ID No:1 protein sequence;
Utilize the method for (+) gamma-lactam enzyme coding gene resolving racemic gamma-lactam preparation (-) gamma-lactam to be:
With raw archeobacteria (Aeropyrum pernix) the NBRC No:100138 of thermophilic spring genome, design primer, upstream primer for masterplate: 5 '-GGAATTC
cATATGgTAACAAGGATAACT-3 '; Downstream primer: 5 '-CCC
aAGCTTcTACTCTGCTAGCTTCTTC-3 '; Carry out polymerase chain reaction (PCR) amplifying target genes, electrophoresis reclaims object fragment; Again described object fragment is connected on T carrier with T4 ligase enzyme, is transformed in E.coli DH5 α, successively dull and stereotyped by blue hickie, containing the dull and stereotyped screening of kantlex; Carry out afterwards single bacterium colony PCR, according to electrophoresis result, select order-checking band, the 37 ℃ of cultivations of successful single bacterium colony of checking order, and carry out carrying in plasmid, obtain the expression vector building; Described expression vector is imported to host cell, with IPTG abduction delivering, obtain recombinant host bacterium thalline, described recombinant host bacterium thalline is carried out to broken (+) gamma-lactam enzyme A-P described in obtaining after centrifugal;
Described recombinant host bacterium thalline is suspended in phosphoric acid buffer, in described phosphoric acid buffer, adds (-) gamma-lactam substrate, react 10～24 hours at 60～120 ℃; Above-mentioned reaction mixture is centrifugal, get supernatant liquor, in described supernatant liquor, add ethyl acetate to extract, obtain described (-) gamma-lactam;
Described (-) gamma-lactam is 2-azabicyclo [2,2,1] heptan-5-alkene-3-ketone.
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| CN103992992A (en) * | 2014-04-09 | 2014-08-20 | 北京化工大学 | Coding gene of (+) gamma-lactamase in Sulfolobus solfataricus P2, and application thereof |
| CN104630194A (en) * | 2015-02-01 | 2015-05-20 | 北京化工大学 | (+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase |
| CN105950595B (en) * | 2016-05-18 | 2019-10-01 | 华东理工大学 | (-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application |
| CN106244572B (en) * | 2016-08-19 | 2019-11-19 | 苏州开元民生科技股份有限公司 | One kind having encoding gene and the application of (+) gamma-lactam enzymatic activity |
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| CN101921742A (en) * | 2010-06-23 | 2010-12-22 | 中国科学院微生物研究所 | (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof |
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| Title |
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| Accession No. NP_147303;Yamazaki, S.等;《GenBank》;20120121 * |
| Yamazaki, S.等.Accession No. NP_147303.《GenBank》.2012, |
| 王建军，郑国钧，吴胜.微生物来源的γ 内酰胺酶研究进展.《微生物学报》.2010,第50卷(第8期),987-994. * |
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