[PubMed] [Google Scholar] 29. racemates. Single enantiomers of dihydropyridines [in most cases, the (4A-914; (iii) enhanced expression of the enzyme in heterologous hosts and in the parent strain; and (iv) comparative biochemical studies with homologous enzymes of protease substrate preference and inhibitory regulation by endogenous proteinaceous protease inhibitors. MATERIALS AND METHODS Genetic manipulations, chemicals, and enzymes. Genetic manipulation for strains and (e.g., isolation of total DNA, transformation, plasmid isolation, colony hybridization, PCR, and DNA sequencing) were performed according to the standard protocols described by Hopwood et al. (12) and Sambrook et al. (19), respectively. Restriction enzymes and T4 DNA ligase were purchased from Takara (Kyoto, Japan). Protease P6, a serine protease from strains were grown for 4 days at 28C in C medium (2% glucose, 2% soluble starch, 2% soybean meal, 0.5% yeast extract, 0.25% NaCl, 0.32% CaCl2??2H2O, 5 g of FeSO4??7H2O per ml, 5 g of MnSO4??5H2O per ml, 5 g of ZnSO4??7 H2O per ml, pH 7.4) with shaking at 220 rpm. Fungal strains were grown at 28C for 3 days in FI medium at the same shaking rate. The culture was centrifuged at 3,000 for 10 min at 4C. A 0.5-ml portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 g of M-801 per ml) in a test tube. The mixture was incubated at 40C for 3 to 24 h. HPLC analysis of biotransformation products. After the pH of the reaction mixture was adjusted to 3.0 with 1 N HCl, the reaction mixture was extracted with an equal volume of ethylacetate. A 200-l portion of the ethylacetate layer was then evaporated to dryness. The residual pellet was dissolved in 500 l of the mobile phase used in the subsequent high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This sample solution (20 l) was applied to an HPLC system equipped with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside diameter]); YMC Co., Ltd., Kyoto, Japan). The column was developed at 50C with 20 mM KH2PO4-methanol (1:1) at a flow rate of 0.8 ml/min. M-801 (retention time, 5.3 min) and its monoester (M-802; retention time, 4.4 min) were detected by UV absorption at 350 nm. P-902 (retention time, 7.0 min) and its monoester (P-903; retention time, 5.1 min) were measured under the same HPLC conditions except that the mobile phase was 60% (in water) methanol-acetic acid (1,000:1) and the column temperature was 35C. The amount of each product was quantitated from the peak area of HPLC based on that of corresponding standard. Enantioselective analysis. To determine the chirality of M-802 by enantioselective chromatography, we used an ULTRON ES-OVM column (150 by 4.6 mm [inside diameter]; Shinwa Chemical Industries, Ltd., Kyoto, Japan). The sample solution (20 l), prepared after a 24-h reaction, was applied to the column, which was developed at 50C with 0.02 M KH2PO4-2-propanol (9:1) at a flow rate of 1 1.0 ml/min. The enantiomers were detected by UV absorption at 350 nm. The chirality of P-903 was determined under the same HPLC conditions except that the mobile phase was 0.02 M KH2PO4-2-propanol (8:2). The retention times of the (4A-914 was cultivated in 1 liter of C medium at 28C for 4 days inside a 3-liter jar fermentor with an aeration rate of 0.5 vol/vol/min. Cells were removed from the growing tradition by centrifugation (8,000 for 5 min. Protease activity for casein was determined by measuring the absorbance at 275 nm of the supernatant liquid. A DHP-A remedy (200 g/ml) was also tested for lipase activity, using a Lipase UV Autotest kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To examine enzyme inhibition by protease inhibitors, 400 l of 250 mM TES [A-914 or protease P6 remedy (3 mg/ml). Phenylmethylsulfonyl fluoride (PMSF) (300 M) or chymostatin (80 M) was added to the combination. M-801 was then added to a final concentration of 300 g/ml. The resulting combination was incubated at 30C for 1 h. The inhibition of the enantioselective hydrolysis was measured from the productivity of M-802 determined by HPLC analysis as explained above. Cloning of an A-914 gene.Compared with protease P6, DHP-A was more alkaliphilic and more thermostable during the course of the enantioselective hydrolysis. instances, the (4A-914; (iii) enhanced expression of the enzyme in heterologous hosts and in the parent strain; and (iv) comparative biochemical studies with homologous enzymes of protease substrate preference and inhibitory rules by endogenous proteinaceous protease inhibitors. MATERIALS AND METHODS Genetic manipulations, chemicals, and enzymes. Genetic manipulation for strains and (e.g., isolation of total DNA, transformation, plasmid isolation, colony hybridization, PCR, and DNA sequencing) were performed according to the standard protocols explained by Hopwood et al. (12) and Sambrook et al. (19), respectively. Restriction enzymes and T4 DNA ligase were purchased from Takara (Kyoto, Japan). Protease P6, a serine protease from strains were cultivated for 4 days at 28C in C medium (2% glucose, 2% soluble starch, 2% soybean meal, 0.5% yeast extract, 0.25% NaCl, 0.32% CaCl2??2H2O, 5 g of FeSO4??7H2O per ml, 5 g of MnSO4??5H2O per ml, 5 g of ZnSO4??7 H2O per ml, pH 7.4) with shaking at 220 rpm. Fungal strains were cultivated at 28C for 3 days in FI medium at the same shaking rate. The tradition was centrifuged at 3,000 for 10 min at 4C. A 0.5-ml portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 g of M-801 per ml) inside a test tube. The combination was incubated at 40C for 3 to 24 h. HPLC analysis of biotransformation products. After the pH of the reaction combination was modified to 3.0 with 1 N HCl, the reaction combination was extracted with an equal volume of ethylacetate. A 200-l portion of the ethylacetate coating was then evaporated to dryness. The residual pellet was dissolved in 500 l of the mobile phase used in the subsequent high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This sample remedy (20 l) was applied to an HPLC system equipped with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside diameter]); YMC Co., Ltd., Kyoto, Japan). The column was developed at 50C with 20 mM KH2PO4-methanol (1:1) at a circulation rate of 0.8 ml/min. M-801 (retention time, 5.3 min) and its monoester (M-802; retention time, 4.4 min) were detected by UV absorption at 350 nm. P-902 (retention time, 7.0 min) and its monoester (P-903; retention time, 5.1 min) were measured under the same HPLC conditions except the mobile phase was 60% (in water) methanol-acetic acid (1,000:1) and the column temperature was 35C. The amount of each product was quantitated from your peak part of HPLC based on that of related standard. Enantioselective analysis. To determine the chirality of M-802 by enantioselective chromatography, we used an ULTRON ES-OVM column (150 by 4.6 mm [inside diameter]; Shinwa Chemical Industries, Ltd., Kyoto, Japan). The sample remedy (20 l), prepared after a 24-h reaction, was applied to the column, which was developed at 50C with 0.02 M KH2PO4-2-propanol (9:1) at a circulation rate of 1 1.0 ml/min. The enantiomers were recognized by UV absorption at 350 nm. The chirality of P-903 was identified under the same HPLC conditions except the mobile phase was 0.02 M KH2PO4-2-propanol (8:2). The retention instances of the (4A-914 was cultivated in 1 GW-1100 liter of C medium at 28C for 4 days inside a 3-liter jar fermentor with an aeration rate of 0.5 vol/vol/min. Cells were removed from the growing tradition by centrifugation (8,000 for 5 min. Protease activity for casein was determined by measuring the absorbance at 275 nm of the supernatant liquid. A DHP-A remedy (200 g/ml) was also tested for lipase activity, using a Lipase UV Autotest kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To.?Fig.2.2. manifestation of the enzyme in heterologous hosts and in the parent strain; and (iv) comparative biochemical studies with homologous enzymes of protease substrate preference and inhibitory rules by endogenous proteinaceous protease inhibitors. MATERIALS AND METHODS Genetic manipulations, chemicals, and enzymes. Genetic manipulation for strains and (e.g., isolation of total DNA, transformation, plasmid isolation, colony hybridization, PCR, and DNA sequencing) were performed according to the standard protocols explained by Hopwood et al. (12) and Sambrook et al. (19), respectively. Restriction enzymes and T4 DNA ligase were purchased from Takara (Kyoto, Japan). Protease P6, a serine protease from strains were cultivated for 4 days at 28C in C medium (2% glucose, 2% soluble starch, 2% soybean meal, 0.5% GW-1100 yeast extract, 0.25% NaCl, 0.32% CaCl2??2H2O, 5 g of FeSO4??7H2O per ml, 5 g of MnSO4??5H2O per ml, 5 g of ZnSO4??7 H2O per ml, pH 7.4) with shaking at 220 rpm. Fungal strains were cultivated at 28C for 3 days in FI medium at the same shaking rate. The tradition was centrifuged at 3,000 for 10 min at 4C. A 0.5-ml portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 g of M-801 per ml) in a test tube. The combination was incubated at 40C for 3 to 24 h. HPLC analysis of biotransformation products. After the pH of the reaction combination was adjusted to 3.0 with 1 N HCl, the reaction combination was extracted with an equal volume of ethylacetate. A 200-l portion of the ethylacetate layer was then evaporated to dryness. The residual pellet was dissolved in 500 l of the mobile phase used in the subsequent high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This sample answer (20 l) was applied to an HPLC system equipped with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside diameter]); YMC Co., Ltd., Kyoto, Japan). The column was developed at 50C with 20 mM KH2PO4-methanol (1:1) at a circulation rate of 0.8 ml/min. M-801 (retention time, 5.3 min) and its monoester (M-802; retention time, 4.4 min) were detected by UV absorption at 350 nm. P-902 (retention time, 7.0 min) and its monoester (P-903; retention time, 5.1 min) were measured under the same HPLC conditions except that this mobile phase was 60% (in water) methanol-acetic acid (1,000:1) and the column temperature was 35C. The amount of each product was quantitated from your peak area of HPLC based on that of corresponding standard. Enantioselective analysis. To determine the chirality of M-802 by enantioselective chromatography, we used an ULTRON ES-OVM column (150 by 4.6 mm [inside diameter]; Shinwa Chemical Industries, Ltd., Kyoto, Japan). The sample answer (20 l), prepared after a 24-h reaction, was applied to the column, which was developed at 50C with 0.02 M KH2PO4-2-propanol (9:1) at a circulation rate of 1 1.0 ml/min. The enantiomers were detected by UV absorption at 350 nm. The chirality of P-903 was decided under the same HPLC conditions except that this mobile phase was 0.02 M KH2PO4-2-propanol (8:2). The retention occasions of the (4A-914 was produced in 1 liter of C medium at 28C for 4 days in a 3-liter jar fermentor with an aeration rate of 0.5 vol/vol/min. Cells were removed from the growing culture by centrifugation (8,000 for 5 min. Protease activity for casein was determined by measuring the absorbance at 275 nm of the supernatant liquid. A DHP-A answer (200 g/ml) was also tested for lipase activity, using a Lipase UV Autotest kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To examine enzyme inhibition by protease inhibitors, 400 l of 250 mM TES [A-914 or protease P6 answer (3 mg/ml). Phenylmethylsulfonyl fluoride (PMSF) (300 M) or chymostatin (80 M) was added to the combination. M-801 was then added to a final concentration of 300 g/ml. The producing combination was incubated at 30C for 1 h. The inhibition of the enantioselective hydrolysis was measured from the productivity of M-802 determined by HPLC analysis as explained above. Cloning of an A-914 gene (A-914 was partially digested with A-914 was constructed according to standard protocols (12), using pIJ702 (15) as a vector and TK24 as a host strain (12). The DNA fragments were inserted into the unique TK24 was transformed with the ligation combination. After drug resistance selection (using.J. expression of the enzyme in heterologous hosts and in the parent strain; and (iv) comparative biochemical studies with homologous enzymes of protease substrate preference and inhibitory regulation by endogenous proteinaceous protease inhibitors. MATERIALS AND METHODS Genetic manipulations, chemicals, and enzymes. Genetic manipulation for strains and (e.g., isolation of total DNA, transformation, plasmid isolation, colony hybridization, PCR, and DNA sequencing) were performed according to the standard protocols explained by Hopwood et al. (12) and Sambrook et al. (19), respectively. Restriction enzymes and T4 DNA ligase were purchased from Takara (Kyoto, Japan). Protease P6, a serine protease from strains were produced for 4 days at 28C in C medium (2% glucose, 2% soluble starch, 2% soybean meal, 0.5% yeast extract, 0.25% NaCl, 0.32% CaCl2??2H2O, 5 g of FeSO4??7H2O per ml, 5 g of MnSO4??5H2O per ml, 5 g of ZnSO4??7 H2O per ml, pH 7.4) with shaking at 220 rpm. Fungal strains were produced at 28C for 3 days in FI medium at the same shaking rate. The culture was centrifuged at 3,000 for 10 min at 4C. A 0.5-ml portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, GW-1100 600 g of M-801 per ml) in a test tube. The combination was incubated at 40C for 3 to 24 h. HPLC analysis of biotransformation products. After the pH of the reaction combination was adjusted to 3.0 with 1 N HCl, the reaction combination was extracted with an equal volume of ethylacetate. A 200-l portion of the ethylacetate layer was then evaporated to dryness. The residual pellet was dissolved in 500 Rabbit Polyclonal to SENP6 l of the mobile phase used in the subsequent high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This sample answer (20 l) was applied to an HPLC system equipped with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside diameter]); YMC Co., Ltd., Kyoto, Japan). The column was developed at 50C with 20 mM KH2PO4-methanol (1:1) at a circulation rate of 0.8 ml/min. M-801 (retention time, 5.3 min) and its monoester (M-802; retention time, 4.4 min) were detected by UV absorption at 350 nm. P-902 (retention time, 7.0 min) and its monoester (P-903; retention time, 5.1 min) were measured beneath the same HPLC conditions except how the cellular phase was 60% (in water) methanol-acetic acidity (1,000:1) as well as the column temperature was 35C. The quantity of each item was quantitated through the peak part of HPLC predicated on that of related regular. Enantioselective analysis. To look for the chirality of M-802 by enantioselective chromatography, we utilized an ULTRON ES-OVM column (150 by 4.6 mm [inside size]; Shinwa Chemical substance Sectors, Ltd., Kyoto, Japan). The test option (20 l), ready after a 24-h response, was put on the column, that was created at 50C with 0.02 M KH2PO4-2-propanol (9:1) at a movement price of just one 1.0 ml/min. The enantiomers had been recognized by UV absorption at 350 nm. The chirality of P-903 was established beneath the same HPLC circumstances except how the cellular stage was 0.02 M KH2PO4-2-propanol (8:2). The retention moments from the (4A-914 was expanded in 1 liter of C moderate at 28C for 4 times inside a 3-liter jar fermentor with an aeration price of 0.5 vol/vol/min. Cells had been taken off the growing tradition by centrifugation (8,000 for 5 min. Protease activity.1980. created for clinical reasons and are utilized as medicines against hypertension and ischemic cardiovascular disease. Although both enantiomers having an asymmetric carbon at the positioning 4 have already been reported to possess different biological actions (14, 20, 30), most 1,4-dihydropyridines are given as racemates. Solitary enantiomers of dihydropyridines [in most instances, the (4A-914; (iii) improved expression from the enzyme in heterologous hosts and in the mother or father stress; and (iv) comparative biochemical research with homologous enzymes of protease substrate choice and inhibitory rules by endogenous proteinaceous protease inhibitors. Components AND METHODS Hereditary manipulations, chemical substances, and enzymes. Hereditary manipulation for strains and (e.g., isolation of total DNA, change, plasmid isolation, colony hybridization, PCR, and DNA sequencing) had been performed based on the regular protocols referred to by Hopwood et al. (12) and Sambrook et al. (19), respectively. Limitation enzymes and T4 DNA ligase had been bought from Takara (Kyoto, Japan). Protease P6, a serine protease from strains had been expanded for 4 times at 28C in C moderate (2% blood sugar, 2% soluble starch, 2% soybean food, 0.5% yeast extract, 0.25% NaCl, 0.32% CaCl2??2H2O, 5 g of FeSO4??7H2O per ml, 5 g of MnSO4??5H2O per ml, 5 g of ZnSO4??7 H2O per ml, pH 7.4) with shaking in 220 rpm. Fungal strains had been expanded at 28C for 3 times in FI moderate at the same shaking price. The tradition was centrifuged at 3,000 for 10 min at 4C. A 0.5-ml part of the supernatant liquid was put into the same level of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 g of M-801 per ml) inside a check tube. The blend was incubated at 40C for 3 to 24 h. HPLC evaluation of biotransformation items. Following the pH from the response blend was modified to 3.0 with 1 N HCl, the reaction blend was extracted with the same level of ethylacetate. A 200-l part of the ethylacetate coating was after that evaporated to dryness. The rest of the pellet was dissolved in 500 l from the cellular phase found in the next high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This test option (20 l) was put on an HPLC program built with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside size]); YMC Co., Ltd., Kyoto, Japan). The column originated at 50C with 20 mM KH2PO4-methanol (1:1) at a movement price of 0.8 ml/min. M-801 (retention period, 5.3 min) and its own monoester (M-802; retention period, 4.4 min) were detected by UV absorption in 350 nm. P-902 (retention period, 7.0 min) and its own monoester (P-903; retention period, 5.1 min) were measured beneath the same HPLC conditions except how the cellular phase was 60% (in water) methanol-acetic acidity (1,000:1) as well as the column temperature was 35C. The quantity of each item was quantitated through the peak part of HPLC predicated on that of related regular. Enantioselective analysis. To look for the chirality of M-802 by enantioselective chromatography, we utilized an ULTRON ES-OVM column (150 by 4.6 mm [inside size]; Shinwa Chemical substance Sectors, Ltd., Kyoto, Japan). The test option (20 l), ready after a 24-h response, was put on the column, that was created at 50C with 0.02 M KH2PO4-2-propanol (9:1) at a movement price of just one 1.0 ml/min. The enantiomers had been recognized by UV absorption at 350 nm. The chirality of P-903 was established beneath the same HPLC circumstances except how the cellular stage was 0.02 M KH2PO4-2-propanol (8:2). The retention moments from the (4A-914 was expanded in 1 liter of C moderate at 28C for 4 times inside a 3-liter jar fermentor with an aeration price of 0.5 vol/vol/min. Cells had been taken off the growing tradition.
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