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With 6His-tagged Pseudomonas fluorescens PfO-1 Pfl_1841 2-methylenebornane synthase In pentane at 30℃; for 12 h; aq. buffer; Enzymatic reaction
General procedure: To 10 mL of assay buffer (50 mM PIPES, 100 mM NaCl, 15 mM MgCl2, 5 mM β-mercaptoethanol and 20percent glycerol, pH 6.7) and 60 μM of FPP, GPP or 2-MeGPP, was added 10 μM of purified Pfl_1841 protein. The enzymatic reaction was overlaid with 10 mL of pentane and incubated at 30 °C for 12 h. Following the incubation, the enzymatic products were extracted with 3.x.10 mL of pentane and the organic extracts were combined, dried over Na2SO4 and concentrated in vacuo to 200 μL for GC-MS analysis
With magnesium chloride at 30℃; for 6h; 0.1M TES buffer, pH=7.0, limonene synthetase;
96 %Chromat.
With Mentha spicata S-limonene synthase; magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With 6His-tagged Pseudomonas fluorescens PfO-1 Pfl_1841 2-methylenebornane synthase; In pentane; at 30℃; for 12.0h;pH 6.7;aq. buffer; Enzymatic reaction;Kinetics;
General procedure: To 10 mL of assay buffer (50 mM PIPES, 100 mM NaCl, 15 mM MgCl2, 5 mM beta-mercaptoethanol and 20% glycerol, pH 6.7) and 60 muM of FPP, GPP or 2-MeGPP, was added 10 muM of purified Pfl_1841 protein. The enzymatic reaction was overlaid with 10 mL of pentane and incubated at 30 C for 12 h. Following the incubation, the enzymatic products were extracted with 3×10 mL of pentane and the organic extracts were combined, dried over Na2SO4 and concentrated in vacuo to 200 muL for GC-MS analysis
With recombinant (+)-cadinene synthase from Gossypium arboreum; magnesium chloride; D,L-dithiothreitol In pentane at 25℃; for 24h; HEPES buffer; Enzymatic reaction;
With recombinant L-tryptophan prenyltransferases/dimethylallyltryptophan synthase from gene SAML0654 of Streptomyces ambofaciens; copper dichloride In aq. acetate buffer at 37℃; for 16h; regiospecific reaction;
With recombinant Cudrania tricuspidata isoliquiritigenin 3'-dimethylallyltransferase; magnesium chloride; In aq. buffer;pH 9;Enzymatic reaction;
General procedure: The assays for the isolation of the enzymatic products (10-15 ml) contained 100 mM Tris-HCl (pH 9.0), 10 mM MgCl2, 1 mM DMAPP, 500 M prenyl acceptors, and 30 mg of protein of the recombinant yeast microsome. The reaction mixtures were incubated at 30 C for 16 h and subsequently extracted with ethyl acetate (20 ml x 5). After evaporation ofthe solvent, the residues were dissolved in methanol and purifiedby reverse-phase semi-preparative HPLC under the conditions described above. The isolated products were subjected to MS and 1Hand 13CNMRspectroscopic analyses, which yielded the following results.
With magnesium chloride; manganese(ll) chloride; In glycerol; at 30℃; for 14h;pH 7.0;Enzymatic reaction;
General procedure: Enzymatic assays: the reaction mixtures were composed of 50 mM Tris buffer (pH 7.0), 10% (v/w) glycerol, 1 mM dithiothreitol, 0.5 mM MnCl2, 20 mM MgCl2, and 28 lM GPP. Purified recombinant proteins were added to the mixtures to a final concentration of 10% (v/v). Completed mixtures were kept at 30 C for 14 h for the enzymatic reaction. The enzymatic assay for each chimera was performed in duplicate. In some cases, the amount of reaction products was very small for short-time reactions and they were not detectable with GC analyses. Therefore, a reaction time of 14 hwas used for all experiments.
With Mentha spicata S-limonene synthase, mutant S454G; magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With Mentha spicata S-limonene synthase, mutant (N345A/L423A/S454A); magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With Mentha spicata S-limonene synthase, mutant N345S; magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With Mentha spicata S-limonene synthase, mutant N345I; magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With Mentha spicata S-limonene synthase, mutant (L423A/S454A); magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With Mentha spicata S-limonene synthase, mutant (C321Y/N345A/L423A/S454A); magnesium chloride; D,L-dithiothreitol In hexane; glycerol at 37℃; for 0.666667h; Enzymatic reaction; chemoselective reaction;
5.4. The GC-MS assay
General procedure: The reaction system in a total volume of 400 μl contains 25 mM Mopso (pH 7.0), 15 mM MgCl2, 1.0 mM DTT, 10% (v/v) glycerol, 68.5 μM GPP and 2 μg S-limonene synthase. The reaction was performed at 37 °C for 40 min. To trap the limonene, 400 ml hexane was added on top of the reaction mixture and vortexed for 30 min. The mixture was centrifuged to separate the organic layer. 1 μl of hexane containing the analytes was analyzed by GC-MS (ThermoTrace GC Ultra& ISQ). The sample was injected into a HP-88 column 100mx250μMx0.20μM thickness (Agilent). Helium (ultra purity) at a flow rate 1.0 ml/min was used as a carrier gas. The oven temperature was first kept constant at 50 °C for 1 min, and then increased to 130 °C at the increment of 3 °C/min, then increased to 220 °C at the increment of 50 C/min, and finally held at thistemperature for 5 min. The injector and transfer line temperatureswere set at 250 C. Monoterpenes were identified from mass spectra and GC retention times by comparing with available authentic standards (S-limonene, α-pinene and β-pinene) and spectra in libraries (α-phellandrene, β-phellandrene and sabinene). The proportion of each product was based on the ratio of the relative peak abundance. The relative activity is calculated by dividing the total peak area for terpene products of one particular enzyme by that of the wild type limonene synthase.
With prenyltransferase FtmPT1 mutant M364G from Aspergillus terreus; calcium chloride In dimethyl sulfoxide; glycerol at 37℃; for 2h; Enzymatic reaction; Overall yield = 54.2 %Chromat.;
With prenyltransferase CdpC2PT mutant T351G from Aspergillus terreus; calcium chloride In dimethyl sulfoxide; glycerol at 37℃; for 16h; Enzymatic reaction;
With prenyltransferase CdpNPT mutant M349G from Aspergillus terreus; calcium chloride In dimethyl sulfoxide; glycerol at 37℃; for 16h; Enzymatic reaction; Overall yield = 40.7 %Chromat.;
With prenyltransferase FgaPT2 double mutant M328G_R244Q from Aspergillus terreus; calcium chloride In dimethyl sulfoxide; glycerol at 37℃; for 2h; Enzymatic reaction;
With aromatic prenyltransferase from Aspergillus terreus; calcium chloride In dimethyl sulfoxide at 37℃; for 16h; Enzymatic reaction; regioselective reaction;
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