Characterization and Immobilization of purified Aspergillus flavipes L-Methioninase: Continuous Production of Methanethiol
Ashraf S. El-Sayed
Faculty of Science
Zagazig University
Egypt
Abstract:
Extracellular and intracellular L-methioninases were purified to the electrophoretic homogeneity from the submerged cultures of Aspergillus flavipes using anion exchange and size exclusion chromatography. By the fourth purification step, the activity of extracellular enzyme was increased by about 1.8 fold than intracellular form, thus, the former was sequentially used for immobilization. Among the tested immobilization methods, polyacrylamide (42.2%), C-alginate (40.9%) and chitin (40.8%) displaying the highest immobilization efficiency. Upon reusing, chitin and polyacrylamide-enzyme conjugates retain 50% of their activities by the fifth cycle. The thermal stability was increased significantly for chitin-enzyme (T1/2 424.1min and Tm 207.5°C) and polyacrylamide-enzyme (T1/2 304.9min and Tm 160.1°C) conjugates, comparing to free enzyme (T1/2 204min and Tm 59.9°C) at 50°C. The thermal inactivation rate was strongly decreased for chitin (3.7×10-3min) and polyacrylamide (4.42×10-3min) enzyme conjugates comparing to free enzyme (8.5×10-3min). The velocity (Vmax) and catalytic efficiency (Kcat) of reaction was slightly decreased for chitin-enzyme (33.8U/mg/min and 12.0S-1) then polyacrylamide-enzyme (33.1U/mg/min and 11.7S-1) conjugates, comparing to the free enzyme (39.6U/mg/min and 14.0S-1). The efficiency of immobilization was greatly improved using 4.0% glutaraldehyde and 41.6/6.3 acrylamide /bisacyylamide, as spacers for chitin and polyacrylamide-enzyme conjugates, respectively, comparing to their controls. Also, incorporation of pyridoxal phosphate, lysine, glutathione, cysteine and dithiothreitol as active site protectants during immobilization drastically improve the catalytic efficiency of immobilized enzyme. Moreover, the activity of immobilized enzyme was increased by 4.5 and 3.5 fold using glutathione plus DDT and glutathione plus methionine, as co-protectants, for chitin and polyacrylamide-enzyme, respectively. Chitin-enzyme gave a plausible operative stability (till 3 cycles) for continuous production of methanethiol under controlled system by trapping on mercuric acetate, that justify the enzymatic synthesis of this compound from the technological point of view. Applying GC-MS and proton NMR analysis, methanethiol salt has identical peaks and chemical structure to the standard compound. Thus, by this work, in addition to the kinetic and catalytic comparative studies for free and immobilized L-methioninase, continuous production of methanethiol by the enzymatic method being a new method, technically
