Characterization and immobilization of purified Aspergillus flavipesl-methioninase: continuous production of methanethiol

Abstract

Aims: To immobilize the purified Aspergillus flavipesl‐methioninase on solid carriers for continuous production of methanethiol with high purity, by the enzymatic methods. Methods and Results: The purified l‐methioninase was immobilized using different methods, and physicochemical and kinetic studies for the potent immobilized enzyme were conducted parallel to the soluble one. The activity of the purified extracellular enzyme was 1·8‐fold higher than intracellular one from submerged cultures of A. flavipes. Among the tested methods, polyacrylamide (42·2%), Ca‐alginate (40·9%) and chitin (40·8%) displayed the highest immobilization efficiency. The thermal inactivation rate was strongly decreased for chitin‐immobilized enzyme (0·222 s−1) comparing to soluble enzyme (0·51 s−1). Enzyme immobilization efficiency was greatly improved using 4·0% glutaraldehyde and 41·6/6·3 (T/C) as spacers for chitin and polyacrylamide‐enzyme conjugates, comparing to their controls. Also the incorporation of lysine, glutathione, cysteine and dithiothreitol as active site protectants significantly enhance the catalytic efficiency of immobilized enzyme. The activity of enzyme was increased by 4·5‐ and 3·5‐fold using glutathione plus DDT and glutathione plus methionine, for chitin and polyacrylamide enzyme, respectively. Conclusion: Chitin enzyme gave a plausible stability till fourth cycle for production of methanethiol under controlled system. Applying GC and HNMR analysis, methanethiol has identical chemical structure to the standard compound. Significance and Impact of the Study: Technically, a new method for continuous production of pure methanethiol, with broad applications, was developed using a simple low expenses method.

Coimmobilization of l‐methioninase and glutamate dehydrogenase: Novel approach for l‐homoalanine synthesis

“…All other chemicals were of analytical grade. GDH is a universal enzyme for the direct amination of 2‐ketobutyrate forming homoalanine . GDH of specific activity 40 U/mg was purchased from Sigma–Aldrich Co.…”

“…l ‐Methioninase was purified from the cultures of A. flavipes JF831014 with specific activity 40 U/mg protein according to previous studies .…”

Biochemical and Pharmacokinetic Properties of Aspergillus flavipes Glutathione-Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor

“…PEG-GHTHase displays fairly resistance to PPG, HA and IA, comparing to native enzyme, suggesting the slight protection by PEG moieties to the surface enzyme amino acids. Practically, biochemical characterization using specific substrates analogues/ inhibitor to explore the modification of enzyme surface amino acids was frequently documented [1,[29][30][31][32].…”

Immobilization and Characterization of Purified Aspergillus flavus Peroxidase Mediated Silver Nanoparticle Synthesis: Peroxidase Surface Reactive Residues are Implemented for Reduction of Silver Ions, More than Its Active Sites

“…To evaluate the potency of peroxidase for bulk synthesis of AgNPs, the enzyme was immobilized on different solid supports. The enzyme was covalently immobilized on chitosan [35] with slight modifications. Briefly, chitosan (1.0 g) was activated by soaking in 100 ml of 0.1 N HCl of 2.5% glutaraldehyde for 3 h at 30°C, then neutralizing the mixture pH to 7.0 by 1 N NaOH.…”

“…The purified peroxidase was entrapped on polyacrylamide and sodium alginate [35] and its potency to synthesize AgNPs was determined. Briefly, 5 ml of acrylamide solution (30%), dissolved in potassium phosphate buffer, 100 µl of ammonium persulfate (10%), mixed thoroughly with 1 ml of peroxidase (35.0 µmol/mg) for 5 min, then 50 µl of TEMED was add.…”