CN104099353A - Regioselective bacterium nitroreductase gene as well as recombinase and application thereof - Google Patents
Regioselective bacterium nitroreductase gene as well as recombinase and application thereof Download PDFInfo
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Abstract
The invention discloses a mutant bacterium nitroreductase gene as well as a recombinase and an application thereof and belongs to the field of enzyme engineering. An Escherichia coli nitroreductase NfsB combined mutant T41L/N71S/F124W can significantly change the regioselectivity of nitro reduction of a multi-nitro substrate and catalyze the specificity of important industrial raw materials, namely, 2,4-dinitrotoluene and 2,4-dimitroanisole to produce corresponding 2-NHOH products through reduction. The method has the advantages of high product purity, mild reaction condition, easiness in enlargement and the like, and enzymes can be designed rationally to change the regioselectivity of nitro reduction according to characteristics of structures of target compounds, so that the method has the good application prospect in production of hydroxylamine compounds.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of gene of saltant type nitroreductase, and the recombinase that contains this gene, and application in nitro locational choice reduction.
Background technology
Aromatic hydroxylamine (having another name called arylamine) is a kind of important organic synthesis intermediate, is widely used in the synthetic of stopper, oxidation inhibitor, agricultural chemicals, medicine, makeup etc.Itself also has very high pharmacology and physiologically active some aromatic hydroxylamine.The preparation of aromatic hydroxylamine at present mainly realizes by selective reduction aromatic nitro compound, and the normal method adopting has Reduction of catalytic hydrogen transfer, metal selected deoxidizing method, electrochemical reducing etc.But these preparation methods are poor to the selectivity ratios of oxyammonia reduction, intermediate aromatic hydroxylamine is converted into rapidly corresponding amine, and easily generates a series of by products such as azo.In addition, chemical reduction oxyammonia is also harsher on reaction conditions, need to use dangerous high-tension unit, inflammable hydrogen, poisonous heavy metal or harmful chemical reagent etc.With respect to traditional chemical preparation process, biological catalysis is except having efficient, special catalytic efficiency, and its maximum advantage is unrivaled chemo-selective and regioselectivity.Meanwhile, the reaction conditions gentleness of biocatalysis can be reacted in the water of normal temperature and pressure, appropriate pH, and this conforms to industrial development targets such as " Sustainable development " " Green Chemistry ".
Nitroreductase is the tenuigenin enzyme that a class depends on FMN or FAD, can utilize NAD (P) H catalysis nitroreduction, finally generates oxyammonia or amino product through nitroso-group intermediate product.Utilize nitroreductase as biological catalyst also original aromatic nitro compound generate the reduction process of corresponding azanol product, more and more receive people's concern.Although most nitroreductases can effectively be realized single nitro-compound or the reduction without many nitro-compounds of locational choice difference, be difficult to realize for the single-minded selective reduction of many nitro-compounds.All location difference of nitro on most many nitro-compounds at present, with important industrial raw materials 2,4-dinitrotoluene (DNT) (24DNT) is example, and nitroreductase can generate 2-oxyammonia-4-nitrotoluene (2HANT) or 4-oxyammonia-2-nitrotoluene (4HANT) by this substrate of catalysis.As 2HANT and the 4HANT of isomers, due to the difference of its oxyammonia group the position of substitution, also different on medicine, agricultural chemicals synthesize and apply.But the oxyammonia mix products that enzyme catalysis generates, because chemical property is close, is difficult to carry out next step separation and purification and obtains single sterling, thereby limited the application of nitroreductase in oxyammonia biosynthesizing.If can pass through the transformation to enzyme molecule, make its specificity in substrate catalytic process generate a certain object reduzate, will greatly reduce later stage separation and purification cost and environmental protection, be conducive to application and the exploitation of nitroreductase in oxyammonia biosynthesizing.
Therefore, find exploitation have different single-minded reduction optionally nitroreductase in hydroxy ammonia compound biosynthesizing, there is significant commercial value and application prospect.
Summary of the invention
For obtaining the nitroreductase of the single-minded locational choice, the present invention is by protein structure and molecular docking model analysis, thereby the amino-acid residue of discovery 124 is by affecting the location of substrate in active centre with the interaction of substrate side-chain radical.If the phenylalanine of 124 (Phe) is sported to tryptophane (Trp), can significantly change the regioselectivity of enzyme.Compared with wild-type nitroreductase, mutant T41L/N71S/F124W is to important industrial raw materials 2, the also reason 4-NHOH product of 4-dinitrotoluene (DNT) (24DNT) and 2,4-dinitroanisol (24DNAN) transfers 2-NHOH product to, realizes 2-NO
2specificity reduction.The implication of T41L described herein, N71S, F124W is respectively: the Threonine of 41 (Thr) is sported to leucine (Leu) (being abbreviated as T41L), the l-asparagine of 71 (Asn) is sported to Serine (Ser) (being abbreviated as N71S), the phenylalanine of 124 (Phe) is sported to tryptophane (Trp) (being abbreviated as F124W).
Physiology substrate and the catalyst mechanism of nitroreductase are still not clear at present, especially be directed to the nitroaromatic of external source, cannot determine the correct location of this substrate in enzymic activity pocket, thus cannot determine with the interactional key amino acid of side-chain radical with and be how to affect the location of substrate in active pocket.Even if simple point mutation has change to a certain degree to the locational choice of enzyme, but be difficult to reach the specificity locational choice.By the analysis to this problem, possible reason is that enzyme is not to be determined by some amino acid to the combination of substrate and location conventionally, but realize by the multiple amino acid actings in conjunction in active pocket.The sudden change in these sites is had to very high risk and uncertainty simultaneously, although can realize to a certain extent the change of the locational choice, may cause that the reduction of enzymatic activity is even lost.For this problem, the present invention, on the basis of simple point mutation, further carries out combinatorial mutagenesis by useful mutational site, investigates the synergy that it is realized selectivity.
The present invention is specifically related to a kind of gene of saltant type nitroreductase, and the recombinant expression vector that contains this gene and recombinant expressed transformant, the preparation method of its recombinase and this recombinase, and this saltant type nitroreductase or contain the application in hydroxy ammonia compound biosynthesizing as catalyzer of the recombinant expressed transformant of this mutated genes.
An aspect of of the present present invention is to provide a kind of gene of saltant type nitroreductase, called after combination mutant T41L/N71S/F124W, and its nucleotide sequence is as shown in SEQ ID NO:1.
Another object of the present invention is to provide the albumen of said mutation type nitroreductase genes encoding, and aminoacid sequence is as shown in SEQ ID NO:2.
Another object of the present invention is to provide the recombinant expression vector and the recombinant expressed transformant thereof that contain this saltant type nitroreductase gene.And genetic engineering bacterium or the transgenic cell line of the DNA sequence dna of the described nitroreduction enzyme mutant of encoding.
Another object of the present invention is to provide a kind of preparation method of the nitroreductase of recombinating, its step comprises: cultivate the above-mentioned recombinant expressed transformant that contains this saltant type nitroreductase gene, from culture, purifying obtains the albumen of mutant bacterial nitroreductase.
Preparation method mentioned above, technical scheme is as follows more specifically for it: encode according to intestinal bacteria (Escherichia coli) nitroreductase NfsB gene NCBI: NC_000913, the mutant primer of design rite-directed mutagenesis, carries out rite-directed mutagenesis taking the cloning vector that carries nitroreductase gene as template and builds mutant; The carrier that maybe can express this enzyme taking pET-28a, as expression vector, maybe can be expressed recombinant plasmid transformed the host cell of this enzyme to e. coli bl21 (DE3) cell, the positive monoclonal of selecting after checking carries out fermentation culture.Obtain saltant type nitroreduction zymoprotein through amalgamation and expression purifying.
Another object of the present invention is to provide albumen or the recombinant expressed transformant of the saltant type nitroreductase genes encoding described in technique scheme, the two as catalyzer at catalysis nitroreduction, in the application in the field such as biosynthesizing and nitro class environmental pollutant biological metabolism of the metabolism of medicine activator, aromatic hydroxylamine compound.Combination mutant T41L/N71S/F124W can efficiently reduce important industrial raw materials 2, and 4-dinitrotoluene (DNT) (24DNT) and 2,4-dinitrobenzene methyl ether (24DNAN) can single-minded selectivity generate 2-NHOH product.
Brief description of the drawings
Fig. 1 .HPLC analyzes relatively wild-type and the regioselectivity of mutant NfsB-T41L/N71S/F124W albumen to 2,4-dinitrotoluene (DNT) (24DNT) reduction.From content shown in figure, mutant NfsB-T41L/N71S/F124W is different from wild-type NfsB to the locational choice of substrate 24DNT reduction, this mutant transfers 2 nitryl groups to by 4 nitro selectivity in 24DNT reduction, generates 2-oxyammonia-4-nitrotoluene (2HANT).Therefore, mutant NfsB-T41L/N71S/F124W can be used as the biosynthesizing of biological catalyst for hydroxy ammonia compound.
Fig. 2 .HPLC analyzes relatively wild-type and the regioselectivity of mutant NfsB-T41L/N71S/F124W albumen to 2,4-dinitroanisol (24DNAN) reduction.From content shown in figure, mutant NfsB-T41L/N71S/F124W is different from wild-type NfsB to the locational choice of substrate 24DNAN reduction, this mutant can specificity 2 nitryl groups in reduction substrate, generate 2-oxyammonia-4-Nitroanisole (2HANAN).Therefore, mutant NfsB-T41L/N71S/F124W can be used as the biosynthesizing of biological catalyst for hydroxy ammonia compound.
Embodiment
Following indefiniteness embodiment can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
The overlapping extension PCR primer sequence that in the present invention, design is used:
nfsB-WT-F：5’-GGAATTC
CATATGGATATCATTTCTGTCGCCTTAAAG-3’；SEQ?ID?NO:3
nfsB-WT-R：5’-G
GAATTCTTACACTTCGGTTAAGGTGATGTT-3’；SEQ?ID?NO:4
nfsB-T41L-F：5’-AGCCCATCCAGCCTGAACTCCCAGCCGTGGCAT-3’；SEQ?ID?NO:5
nfsB-T41L-R：5’-CTGGGAGTTCAGGCTGGATGGGCTGTATTGCA-3’SEQ?ID?NO:6
nfsB-N71S-F：5’-ATGTGTTCAGCGAACGTAAAATGCTTGATG-3’；SEQ?ID?NO:7
nfsB-N71S-R：5’-ATTTTACGTTCGCTGAACACATAATTACCGGCAG-3’SEQ?ID?NO:8
nfsB-F124W-F：5’-TCGCAAGTTCTGGGCTGATATGCACCGTAAAGATC-3’；SEQ?ID?NO:9
nfsB-F124W-R：5’-TATCAGCCCAGAACTTGCGACCTTTATCGTTCG-3’SEQ?ID?NO:10
Embodiment 1 mutant bacterial reductase enzyme NfsB-T41L/N71S/F124W gene obtains and expression vector establishment
1. the clone of wild-type nfsB gene and cloning vector build
NfsB gene order in the E.coli K12 bacterial strain (Invitrogen) providing according to NCBI, and design primer (nfsB-WT-F:5 '-GGAATTC
cATATGgATATCATTTCTGTCGCCTTAAAG-3 ', nfsB-WT-R:5 '-G
gAATTCtTACACTTCGGTTAAGGTGATGTT-3 '), wherein underscore part represents the gene order that restriction enzyme site is corresponding.Utilizing the E.coli K12 genomic dna extracting is masterplate, carries out pcr amplification.Get 10 μ l PCR reaction product and run agarose gel electrophoresis checking, the about 650bp of electrophoretic band and nfsB gene are in the same size.Use TaKaRaAgarose Gel DNA Purification Kit Ver.3.0 to cut glue and reclaim above-mentioned electrophoresis object band, then process and reclaim product with EcoR I/Nde I double digestion, use TaKaRa DNA Fragment Purification Kit Ver.3.0 to be purified, use afterwards the ligase enzyme in DNA Ligation Kit Ver.3.0, by the object product obtaining after purifying with after pET28a (+) empty carrier of EcoR I/Nde I double digestion processing is connected, heat shock is converted in E.coli DH5 α competent cell, spread plate, 37 DEG C of incubated overnight.Selecting positive bacterium colony spends the night and extracts plasmid and send TaKaRa order-checking to confirm consistent with nfsB gene (Gene ID:945778) sequence after 37 DEG C of cultivations, checking gene clone success, and by its called after wild-type nfsB gene, and by the positive bacterium colony of its correspondence and recombinant plasmid called after clone bacterium E.coliDH5 α-nfsB-WT and plasmid pET28a-nfsB-WT respectively.
2. the gene of saltant type NfsB-T41L/N71S/F124W obtains and expression vector establishment
Incubated overnight E.coliDH5 α-nfsB-WT clones bacterium, extracts pET28a-nfsB-WT recombinant plasmid, builds mutant carrier pET28a-nfsB-T41L/N71S/F124W as template.
(1), first taking plasmid pET28a-nfsB-WT as template, use primer nfsB-WT-F and nfsB-F124W-R:5 '-TATCAGC
cCAgAACTTGCGACCTTTATCGTTCG-3 ' carries out a PCR reaction, called after nfsB-F124W-1.Taking plasmid pET28a-nfsB-WT as template, use primer nfsB-WT-R and nfsB-F124W-F:5 '-TCGCAAGTTC equally
tGGgCTGATATGCACCGTAAAGATC-3 ' carries out secondary PCR reaction, called after nfsB-F124W-2.Wherein in primer nfsB-F124W-F and nfsB-F124W-R underscore mark be 124 gene orders corresponding to amino acids mutational site.Use TaKaRaMiniBestAgarose Gel DNA Extraction Kit Ver.3.0, cut glue for twice PCR product and reclaim purifying.To reclaim product nfsB-F124W-1 and nfsB-F124W-2 as template, use primer nfsB-WT-F and nfsB-WT-R to carry out overlapping extension PCR for the third time, PCR product uses TaKaRa DNA Fragment Purification Kit Ver.3.0 to reclaim purifying, its called after nfsB-F124W.
(2) nfsB-F124W obtaining taking step (1), as template, uses primer nfsB-WT-F and nfsB-T41L-R:5 '-CTGGGAGTT
cAGgCTGGATGGGCTGTATTGCA-3 ' carries out a PCR reaction, called after nfsB-T41L/F124W-1.Taking nfsB-F124W as template, use primer nfsB-WT-R and nfsB-T41L-F:5 '-AGCCCATCCAGC equally
cTGaACTCCCAGCCGTGGCAT-3 ' carries out secondary PCR reaction, called after nfsB-T41L/F124W-2.Wherein in primer nfsB-T41L-F and nfsB-T41L-R underscore mark be 41 gene orders corresponding to amino acids mutational site.Use TaKaRaMiniBestAgarose Gel DNA Extraction Kit Ver.3.0, cut glue for twice PCR product and reclaim purifying.To reclaim product nfsB-T41L/F124W-1 and nfsB-T41L/F124W-2 as template, use primer nfsB-WT-F and nfsB-WT-R to carry out overlapping extension PCR for the third time, PCR product uses TaKaRa DNA Fragment Purification Kit Ver.3.0 to reclaim purifying, its called after nfsB-T41L/F124W.
(3) nfsB-T41L/F124W obtaining taking step (2), as template, uses primer nfsB-WT-F and nfsB-N71S-R:5 '-ATTTTACGTTC
gCTgAACACATAATTACCGGCAG-3 ' carries out a PCR reaction, called after nfsB-T41L/N71S/F124W-1.Taking nfsB-T41L/F124W as template, use primer nfsB-WT-R and nfsB-N71S-F:5 '-ATGTGTTC equally
aGCgAACGTAAAATGCTTGATG-3 ' carries out secondary PCR reaction, called after nfsB-T41L/N71S/F124W-2.Wherein in primer nfsB-N71S-F and nfsB-N71S-R underscore mark be 71 gene orders corresponding to amino acids mutational site.Use TaKaRaMiniBestAgarose Gel DNA Extraction Kit Ver.3.0, cut glue for twice PCR product and reclaim purifying.To reclaim product nfsB-T41L/N71S/F124W-1 and nfsB-T41L/N71S/F124W-2 as template, use primer nfsB-WT-F and nfsB-WT-R to carry out overlapping extension PCR for the third time, PCR product uses TaKaRa DNA Fragment Purification Kit Ver.3.0 to reclaim purifying, obtain mutant gene, its called after nfsB-T41L/N71S/F124W gene, its sequence information is SEQ ID NO:1.
(4) the mutant gene SEQ ID NO:1 obtaining and the unloaded Nde I/EcoR I that uses of pET28a (+) are carried out respectively to double digestion, use TaKaRaAgarose Gel DNA Purification Kit Ver.3.0 to cut glue and reclaim above-mentioned electrophoresis object band, use afterwards the ligase enzyme in DNA Ligation Kit Ver.3.0, by the object product obtaining after purifying with after pET28a (+) carrier of EcoR I/Nde I double digestion processing is connected, heat shock is converted in E.coli DH5 α competent cell, spread plate, 37 DEG C of incubated overnight.Selecting positive bacterium colony spends the night and extracts plasmid and send TaKaRa order-checking to confirm consistent with SEQ ID NO:1 sequence after 37 DEG C of cultivations, the success of checking expression vector establishment, and by the positive bacterium colony of its correspondence and recombinant plasmid called after clone bacterium E.coliDH5 α-nfsB-T41L/N71S/F124W and plasmid pET28a-nfsB-T41L/N71S/F124W.
The expression and purification of embodiment 2. bacterium nitroreduction enzyme mutant NfsB-T41L/N71S/F124W
1. the abduction delivering of nitroreduction enzyme mutant NfsB-T41L/N71S/F124W
Recombinant plasmid pET28a-nfsB-T41L/N71S/F124W heat shock is transformed in intestinal bacteria E.coli BL21 (DE3) competent cell (Invitrogen) to spread plate, 37 DEG C of incubated overnight.Picking positive colony, and its called after is expressed to bacterium E.coliBL21 (DE3)-nfsB-T41L/N71S/F124W, be inoculated in (containing 50 μ g/ml Kana) in LB liquid nutrient medium 37 DEG C of shaken overnight.Seed culture fluid is forwarded to (containing 50 μ g/ml Kana) in 100ml LB enlarged culturing base with 1% inoculum size, and 37 DEG C of shaking tables are cultured to OD
600at 0.4-0.6.Adding final concentration is 0.5mM IPTG abduction delivering 6h at 30 DEG C.After cultivation finishes, fermented liquid is collected thalline in the centrifugal 10min of 5000r/min.
2. the purifying of nitroreduction enzyme mutant NfsB-T41L/N71S/F124W
Collection thalline carries out resuspended with 20mM phosphate buffered saline buffer (pH7.4 contains 500mMNaCl), utilize the broken instrument of high-pressure homogenization to carry out at low temperatures fragmentation, and the centrifugal 5min of 12000r/min collects supernatant.
Sample separation purifying all uses
purifier protein flash chromatography system (GE Healthcare) completes, and adopts GE Healthcare FF HisTrap chromatography column (being Ni post).First use buffer A (20mM NaH
2pO4,0.5MNaCl, pH7.4) balance Ni post, the albumen supernatant sample that fragmentation is collected afterwards passes through Ni post with the flow velocity of 1ml/min, makes target protein be incorporated into Ni post.After ultraviolet baseline is steady, use buffer B (elution buffer:20mM NaH2PO4,0.5MNaCl, 250mM imidazoles, pH7.4) wash-out, gradient is followed successively by 20mM, 100mM, 250mM imidazoles, each gradient elution volume is 20 column volumes.Be in charge of the effluent liquid of collecting each gradient elution, every pipe sample carried out respectively to protein content, vigor and SDS-PAGE and analyze.
3. the SDS-PAGE electrophoretic analysis of nitroreduction enzyme mutant NfsB-T41L/N71S/F124W and Western-Blotting checking
Utilize molecular weight and the purity thereof of SDS-PAGE electrophoretic analysis saltant type nitroreductase NfsB-T41L/N71S/F124W, contrast Marker selects lower molecular weight standard protein.After electrophoresis finishes, dye more than 3 hours with coomassie brilliant blue R_250, with destainer (taking ethanol: acetic acid: the volume ratio of water as 1:2:17 formulated) concussion decolouring, observe electrophoresis result.
Electrophoresis result finds there is a significantly induction band in molecular weight 27kDa left and right.Induce the corresponding albumen of band to obtain enrichment at 250mM imidazoles elution fraction simultaneously, tentatively judge that this albumen is mutant protein NfsB-T41L/N71S/F124W, its sequence information is SEQ ID NO:2.
For transferring film instrument, adopt semidrying transferring film, transferring film damping fluid is 48mMTris, 39mM glycine, 20% methyl alcohol, 0.037%SDS, concrete steps are as follows:
Sds page is taken off, be placed in transferring film damping fluid balance, cut out the pvdf membrane that a piece size is identical, be positioned in 100% methyl alcohol and activate 10s, then film is washed to methyl alcohol remaining on film with deionized water, be placed in transferring film damping fluid; Press filter paper-pvdf membrane-gel-filter paper, sequence from low to uper part assembling, prepares transferring film; By complete the assembling of transferring film instrument, constant voltage 18V is set, transferring film 2 hours;
Sealing: the pvdf membrane having turned is immersed in the TBST damping fluid of the skim-milk that contains 5% to sealing 1h in (20mMTris-HCl, 150mMNaCl, 0.05% Tween20);
Primary antibodie is coated: the pvdf membrane having sealed is soaked in containing (containing anti-His antibody, 1:3000, Invitrogen, 3% skim-milk) in primary antibodie TBST coating buffer 1 hour; Wash once every 10min with TBST damping fluid, wash 6 times, remove non-specific binding antibody;
Two is anti-coated: the pvdf membrane in upper step is immersed in the skim-milk that contains mountain sheep anti mouse IgG/ horseradish enzymic-labelled antibody and 3% TBST damping fluid in, vibrate 1 hour; Wash once every 10min with TBST damping fluid, wash 6 times,, remove the two anti-of non-specific binding;
Wash once every 10min with TBST damping fluid, wash 6 times, add chromophoric substrate reaction 3-5min, exposure, develops, photographic fixing.
Western-Blotting result shows that in IPTG induction component, molecular weight 27kDa place protein band is target protein band, and this albumen after Ni column separating purification in the lower concentration and separation that obtains of 250mM imidazoles, purity reaches more than 90%.
The determination of activity of embodiment 3. bacterium nitroreduction enzyme mutant NfsB-T41L/N71S/F124W
The recombinant protein that 250mM imidazoles is eluted, mixes according to the ratio of molar concentration rate albumen: FMN=1:10, places in 4 DEG C of refrigerators and hatches 2 hours.Hatch after end, by recombinant protein desalination to 20mMTris-HCl damping fluid (pH7.0), to carry out the experiments such as follow-up protein-active detection.
1.HPLC analyzes high performance liquid chromatography for regioselectivity (HPLC) Agilent 1200 of mutant NfsB-T41L/N71S/F124W reductase 12 4DNT and analyzes the reduzate of mutant to 24DNT.Taking 24DNT as substrate, detect reduction efficiency and the reduzate ratio of mutant.In HPLC enzymic catalytic reaction system, contain: 0.1mM24DNT, 0.2mM NADH, 200nM mutant enzyme, reaction buffer is 20mMTris-HCl pH7.0, room temperature reaction 5-15 minute.Because 24DNT and reaction product thereof add thermally labile, so extract with termination reaction the centrifugal 10min of 12000 × g by equal-volume ethyl acetate.Getting upper strata ethyl acetate analyzes for HPLC.HPLC analytical column is selected the anti-phase hydrophobic chromatography post of C18, particle diameter 5 μ m, and 4.6mm × 250mm, mobile phase composition is methyl alcohol and aqueous systems, and flow velocity is 0.8ml/min, and sampling volume is 10 μ l, detects by UV-detector.HPLC analysis system is 20% methyl alcohol balance, after sample introduction through 50% methyl alcohol Gradient elution 15min, the methyl alcohol 10min linear elution of 50%-90%, detection wavelength is 254nm, 20 DEG C of column temperatures.Two kinds of oxyammonia product retention time are respectively: R
4-NHOH=13.8min, R
2-NHOH=14.1min.
As shown in Figure 1, mutant NfsB-T41L/N71S/F124W is to important industrial raw materials 2 for experimental result, and the also reason 4-NHOH product of 4-dinitrotoluene (DNT) (24DNT) transfers 2-NHOH product to.
2.HPLC analyzes high performance liquid chromatography for regioselectivity (HPLC) Agilent 1200 of mutant NfsB-T41L/N71S/F124W reductase 12 4DNAN and analyzes the reduzate of mutant to 24DNAN.Taking 24DNAN as substrate, detect reduction efficiency and the reduzate ratio of mutant.In HPLC enzymic catalytic reaction system, contain: 0.1mM24DNAN, 0.2mM NADH, 200nM mutant enzyme, reaction buffer is 20mMTris-HCl pH7.0, room temperature reaction 15-30 minute, uses equal-volume ethyl acetate to extract with termination reaction, the centrifugal 10min of 12000 × g.Getting upper strata ethyl acetate analyzes for HPLC.HPLC analytical column is selected the anti-phase hydrophobic chromatography post of C18, particle diameter 5 μ m, and 4.6mm × 250mm, mobile phase composition is acetonitrile and aqueous systems, and flow velocity is 0.8ml/min, and sampling volume is 10 μ l, detects by UV-detector.HPLC analysis system is 20% acetonitrile balance, the acetonitrile 20min linear elution of 40%-80% after sample introduction, and detection wavelength is 263nm, 20 DEG C of column temperatures.Two kinds of oxyammonia product retention time are respectively: R
4-NHOH=7.1min, R
2-NHOH=8.6min.
As shown in Figure 2, mutant NfsB-T41L/N71S/F124W can realize important industrial raw materials 2 experimental result, the specificity reduction of 4-dinitroanisol (24DNAN), and reduzate transfers 2-NHOH product to by 4-NHOH product.
3. the kinetic determination of nitroreduction enzyme mutant
All kinetic constants be all with full wavelength scanner fluorescence microplate reader (
thermo Fisher Scientific) by the method for continuous measuring point in 37 DEG C of Constant Temperature Detection.Nitroreductase kinetic measurement system: cumulative volume 200 μ l, contain 60 μ M NADH, the substrate to be measured of a series of different concns and appropriate enzyme in the damping fluid of 20mMTris-HCl (pH7.0).Carry out with reaction, by detecting the consumption of NADH under 340nm, and then calculate base consumption speed of reaction.Every reduction 1mol nitryl group, need to consume the NADH molecule of 2mol.All dynamic experiments at least repeat 3 times, and data are processed and drawn data and limit of error with nonlinear fitting software Origin8.5.
Table 1. wild-type and mutant NfsB-T41L/N71S/F124W be to substrate 2, the kinetic constant of 4-dinitrotoluene (DNT) (24DNT) and 2,4-dinitroanisol (24DNAN).
From data analysis in table, catalytic activity and the wild-type of mutant NfsB-T41L/N71S/F124W to substrate 24DNT is similar, and compares and improved 3 times compared with wild-type for the catalytic activity of 24DNAN.Therefore, mutant NfsB-T41L/N71S/F124W, in the selectively changing of feasible region, successfully keeps and has improved the catalytic activity of enzyme, can be used as biological catalyst synthetic for the specificity reduction of hydroxy ammonia compound.
Claims (6)
1. a locational choice bacterium nitroreduction mutant gene, its nucleotide sequence is as shown in SEQ ID NO:1.
2. the coded protein of locational choice bacterium nitroreduction mutant gene as claimed in claim 1, has the aminoacid sequence shown in SEQ ID NO:2.
3. one kind comprises the recombinant expression vector of gene as claimed in claim 1.
4. a recombinant expressed transformant that comprises recombinant expression vector as claimed in claim 3.
5. a preparation method for locational choice bacterium nitroreductase, is characterized in that: cultivate recombinant expressed transformant as claimed in claim 4, from culture, purifying obtains the albumen of mutant bacterial nitroreductase.
Protein as claimed in claim 2 or recombinant expressed transformant as claimed in claim 4 as catalyzer at catalysis nitroreduction, in the biosynthesizing of the metabolism of medicine activator, aromatic hydroxylamine compound and the application in nitro class environmental pollutant biological metabolism field.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050013808A1 (en) * | 2001-08-21 | 2005-01-20 | Grove Jane Isabel | Nitroreductase enzymes |
| US20120244138A1 (en) * | 2011-03-24 | 2012-09-27 | Academia Sinica | Antidotes for nitrobenzodiazepines |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050013808A1 (en) * | 2001-08-21 | 2005-01-20 | Grove Jane Isabel | Nitroreductase enzymes |
| US20120244138A1 (en) * | 2011-03-24 | 2012-09-27 | Academia Sinica | Antidotes for nitrobenzodiazepines |
Non-Patent Citations (3)
| Title |
|---|
| MANSOOREH JABERIPOUR 等: "Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations", 《BIOCHEMICAL PHARMACOLOGY》 * |
| SHIUAN-WOEI LINWU 等: "Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis", 《BIOCHEMICAL PHARMACOLOGY》 * |
| 白敬 等: "细菌硝基还原酶的结构与催化机制", 《生命的化学》 * |
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| CN104099353B (en) | 2017-01-11 |
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