A strain improvement program was initiated to increase the yield of extracellular phytase (EC 184.108.40.206) in Aspergillus ficuum NRRL 3135. A minimal plating medium containing 2.99 g liter-1 a-ketoglutaric acid was developed to decrease and stabilized conidiogenesis time and facilitate the isolation of single colonies. Exposure of A. ficuum conidia to 0.5 mg ml-1 N-methyl-N’-nitro-N-nitrosoguanidine (NTG) for 15 min at pH 6.5 caused a one log kill and an increase in the mutation rate of 800-fold. Treatment of A. ficuum conidia with 46 ergs mm-2 sec -1 UV radiation for 30 sec resulted in a one log kill and a 100,000-fold increase in mutation rate. Mutants which overproduce extracellular acid phosphatase(s) were tentatively identified in a plate assay by histochemical staining with 0.1 g ml-1 a-naphthyl acid phosphate and 0.04 g ml-1 fast black K-salt in 0.2 M acetate buffer pH 4.8. Confirmation of extracellular acid phosphatase overproduction was by analysis of shaker flask fermentation broths via colorimetric enzyme assays with 0.04 M p-nitrophenyl phosphate and 2.5 µM sodium phytate as substrates. A UV derived mutant had a two-fold decrease in conidiogenesis time and a 17% increase in acid phosphatase activity. Further characterization of this mutant by SDS-PAGE electrophoresis and inhibition studies using 1 mM fluoride showed that the apparent increase in enzyme activity was caused by hyperproduction or hypersecretion of a nonspecific acid phosphate (EC 220.127.116.11).
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Wodzinski, Rudy J.
Master of Science (M.S.)
College of Arts and Sciences
Length of Campus-only Access
Masters Thesis (Open Access)
Utt, Eric A., "The Strain Improvement of Aspergillus Ficuum NRRL 3135" (1987). Retrospective Theses and Dissertations. 5043.
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