Tag Archives: Purification

Purification and Characterization of Psuedomonas Aeruginosa Iz Cellulase Grown On Corn Cobs (Published)

Partial purification of crude Psuedomonas aeruginosa IZ cellulase enzyme was carried out by ammonium sulfate fractionation (85%). The specific activity of cellulase increased 493.3% (from 0.15 U/ml protein in the crude enzyme to 0.89 U/ml protein after ammonium sulfate treatment) while the unit protein was decreased 21.9% (from 48.5 mg/ml in the crude enzyme to 37.9 mg/ml after ammonium sulfate treatment) with activity preservation and a purification fold of 5.93.  Further purification of the partially purified enzyme was achieved using anion exchange chromatography on DEAE sephadex A-50. The cellulase activity was enriched after this step of purification; the specific activity increased 301.1 % (from 0.89U/ml protein after ammonium sulfate treatment to 3.57 U/ml protein after anion exchange chromatography) with a purification fold of 23.8. The protein was reduced by 56.99 % (from 37.9 after ammonium sulfate treatment to 16.3 after anion exchange chromatography).  The highest active cellulase fractions obtained from anion exchange chromatography on DEAE sephadex A-50 were loaded on a Sephadex G-100 column for Gel filtration chromatography. The specific activity of cellulase was further increased by 85.71 % (3.57 U/ml protein after anion exchange chromatography to 6.63 U/ml after gel filtration) with purification factor fold of 44.2 and protein decreased by 39.26 % (from 16.3mg/ml after anion exchange chromatography to 9.9 mg/ml after gel filtration chromatography). The optimum pH and incubation temperature for the purified Psuedomonas aeruginosa IZ cellulase enzyme were 6.5 and 35˚C respectively. The enzyme kinetics studies indicated that the enzyme activity is dependent on the concentration of the substrate. When the relation between the enzyme activity and the substrate concentration was treated by Lineweaver-Burk analysis, it was found that Km and Vmax were 4 mg/ml and 7.2 U/ml respectively.

Keywords: Characterization, Psuedomonas aeruginosa IZ, Purification, cellulase, corn cobs

Purification and characterization of Psuedomonas aeruginosa IZ cellulase grown on corn cob (Published)

Partial purification of crude Psuedomonas aeruginosa IZ cellulase enzyme was carried out by ammonium sulfate fractionation (85%). The specific activity of cellulase increased 493.3% (from 0.15 U/ml protein in the crude enzyme to 0.89 U/ml protein after ammonium sulfate treatment) while the unit protein was decreased 21.9% (from 48.5 mg/ml in the crude enzyme to 37.9 mg/ml after ammonium sulfate treatment) with activity preservation and a purification fold of 5.93.  Further purification of the partially purified enzyme was achieved using anion exchange chromatography on DEAE sephadex A-50. The cellulase activity was enriched after this step of purification; the specific activity increased 301.1 % (from 0.89U/ml protein after ammonium sulfate treatment to 3.57 U/ml protein after anion exchange chromatography) with a purification fold of 23.8. The protein was reduced by 56.99 % (from 37.9 after ammonium sulfate treatment to 16.3 after anion exchange chromatography).  The highest active cellulase fractions obtained from anion exchange chromatography on DEAE sephadex A-50 were loaded on a Sephadex G-100 column for Gel filtration chromatography. The specific activity of cellulase was further increased by 85.71 % (3.57 U/ml protein after anion exchange chromatography to 6.63 U/ml after gel filtration) with purification factor fold of 44.2 and protein decreased by 39.26 % (from 16.3mg/ml after anion exchange chromatography to 9.9 mg/ml after gel filtration chromatography). The optimum pH and incubation temperature for the purified Psuedomonas aeruginosa IZ cellulase enzyme were 6.5 and 35˚C respectively. The enzyme kinetics studies indicated that the enzyme activity is dependent on the concentration of the substrate. When the relation between the enzyme activity and the substrate concentration was treated by Lineweaver-Burk analysis, it was found that Km and Vmax were 4 mg/ml and 7.2 U/ml respectively.

 

Keywords: Characterization, Psuedomonas aeruginosa IZ, Purification, cellulase, corn cobs

PURIFICATION AND CHARACTERIZATION OF STENOTROPHOMONAS SP.FZ L-ASPARAGINASE UNDER SOLID STATE FERMENTATION (Published)

L-asparaginase has emerged as one of the most important clinically used enzymes as it exhibits chemotherapeutic potential in treatment of acute lymphoblastic leukimia and lymphosacroma. A novel bacterium L-asparaginase producer was isolated from Lebanese soil and was identified as Stenotrophomonas sp.FZ using 16srRNA. The enzyme was produced under solid state fermentation using the wheat bran as carbon and nitrogen source The enzyme was partially purified by acetone precipitation with 73.32 % yield and a purification factor of 6.32 fold. Further purification includes gel filtration chromatoghraphy on Sephadex G-75 and C-25 ion exchange chromatogrraphy with final purification factor of 329.061 fold and 54.312 yield%. The total protein was reduced by 99.83% and the specific activity was increased to be 1.6124IU/mgX1000. The maximum enzyme activity was recorded at pH 7 and 35°C with a linear relationship concerning the increase in enzyme concentration. The effect of substrate concentration showed a progressive increase in the enzyme activity in a concentration dependent manner till it reaches a plateau where saturation was reached. The kinetics parameters(Km and Vmax ) of Stenotrophomonassp L-asparaginase production were 96.71 mg/ml and 3333.33 umol/ml/min respectively. The theapeutic potential of this enzyme is well established.

Keywords: Characterization, L-Asparaginase., Purification, Stenotrophomonassp, Therapeutic Potential

PARTIAL PURIFICATION AND CHARACTERIZATION OF ENDO-Β-GLUCANASES OF TWO NIGERIAN MALTED MAIZE VARIETIES (Published)

In this study, two Nigerian maize varieties (Farz 23 yellow and Farz 34 white) were malted at controlled experimental variables to determine the optimum conditions for the development of β-glucanases. The independent variable employed were steeping time, germination time and kilning temperature; and the measured (dependent variables) were diastatic power (DP), cold water extract (CWE) and hot water extract (HWE). Some properties of the two varieties were compared with those of sorghum (SK 5912). The properties of the grains were obtained as 1000-corn weight, (W) 241g and (Y) 248g; moisture content (%) 13.2, 12.8; germinative energy (%) 96, 92; germinative capacity (%) 99, 96; water sensitivity (%) 83, 82; broken kernel (%) 0.9, 1.1; protein (N×6.25%) 8.65, 9.02; and fat (ether extract, %) 3.70 and 4.14. The β-glucanases purified 2.59 and 0.56 folds for the yellow and white varieties respectively by a combination of 5M sucrose fractionation, ion exchange on Q-Sepharose and gel filtration on Sephadex G-200; with a yield of 0.8% (yellow) and 7.6% (white). The specific activity for the yellow maize enzyme was 0.312µ/mg and 0.381µ/mg for the white. The optimal condition for the glucanase activity for the white variety were 50oC and pH 5.0 and 7.0, while maximum stability was achieved at 40oC, pH 5.0 and 7.5 (16h); and for the yellow 60oC and pH 5.0 and maximum stability at 40oC, pH 6.0 (16h). The purified enzyme of the white variety was appreciably activated by Co2+, Mn2+ and Zn2+ gave inhibitory effects. The yellow variety glucanase activity was only enhanced by Mn2+ and fairly by Co2+. The yellow variety glucanase displayed remarkable wide substrate specificity and rapidly hydrolyzed amylose, amylopectin and starch. On substrate concentration the white maize enzyme indicated substrate enhancement on both Sigma cell type 20 and CMC. The Km values as obtained from Lineweaver-Burk plots for the white maize glucanase were 0.119 and 0.102mg/ml for CMC and Sigma cell respectively, and 0.072 and 0.0451mg/ml in same order for the yellow maize glucanase. Their corresponding Vmax were 3.7 and 3.5mg/ml/min for the white maize enzyme and 4.0 and 9.0mg/ml/min protein for the yellow maize enzyme. The very low Km values Sigma cell type 20 and CMC for the yellow indicates a high affinity of the yellow glucanase to the substrates. On overall assessment, the glucanases presents a promising application in the mashing conditions of the brew house.

Keywords: Characterization, Maize, Malting, Purification, β-glucanase

PURIFICATION AND CHARACTERIZATION OF L-ASPARAGINASE FROM A SOIL ISOLATE UNDER SOLID STATE FERMENTATION (Published)

L-asparaginase has emerged as one of the most important clinically used enzymes as it exhibits chemotherapeutic potential in treatment of acute lymphoblastic leukimia and lymphosacroma. Soil microbial isolates were screened for potential producers of L-asparaginase using a phenol red indicator growth medium and the microbe producing the largest hydrolysis zone was selected. The isolate was characterized by biochemical tests and was found to belongs to Stenotrophomonas sp. The enzyme was partially purified by acetone precipitation with 73.32 % yield and a purification factor of 6.32 fold. Further purification includes gel filtration chromatoghraphy on Sephadex G-75 and C-25. The maximum enzyme activity was recorded at pH 7 and 35°C with a linear relationship concerning the increase in enzyme concentration. The effect of substrate concentration showed a progressive increase in the enzyme activity in a concentration dependent manner till it reaches a plateau where saturation was reached. The kinetics parameters (Km and Vmax ) of Stenotrophomonas sp L-asparaginase production were 96.71 mg/ml and 3333.33 umol/ml/min respectively.

Keywords: Characterization, L-Asparaginase., Purification, Stenotrophomonas sp

PURIFICATION AND CHARACTERIZATION OF STENOTROPHOMONASSP.FZ L-ASPARAGINASE UNDER SOLID STATE FERMENTATION (Published)

L-asparaginase has emerged as one of the most important clinically used enzymes as it exhibits chemotherapeutic potential in treatment of acute lymphoblastic leukimia and lymphosacroma. A novel bacterium L-asparaginaseproducerwas isolated from Lebanesesoil andwas identified asStenotrophomonas sp.FZ using 16srRNA. The enzyme was produced under solid state fermentation using the wheat bran as carbon and nitrogen source The enzyme was partially purified by acetone precipitation with 73.32 % yield and a purification factor of 6.32 fold. Further purification includes gel filtration chromatoghraphy on Sephadex G-75 and C-25 ion exchange chromatogrraphy with final purification factor of 329.061 fold and 54.312 yield%. The total protein was reduced by 99.83% and the specific activity was increased to be 1.6124IU/mgX1000. The maximum enzyme activity was recorded at pH 7 and 35°C with a linear relationship concerning the increase in enzyme concentration. The effect of substrate concentration showed a progressive increase in the enzyme activity in a concentration dependent manner till it reaches a plateau where saturation was reached. The kinetics parameters(Km and Vmax ) of Stenotrophomonassp L-asparaginase production were 96.71 mg/ml and 3333.33 umol/ml/min respectively. The theapeutic potential of this enzyme is well established.

Keywords: Characterization, L-Asparaginase., Purification, Stenotrophomonassp, Therapeutic Potential