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The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

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The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH~-salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NHn)H2PO4 as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70°C and showed similar thermal stability. By incubation without substrate at 60 ° C, 90% of the activity was still present after 5 h. At 70 ° C, 50% of the activity was retained after 30 min incubatiom

Uptake of phenylacetic acid, the side‐chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54‐1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high‐yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid‐derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250–3500 μM), and in the high‐yielding strain at low phenylacetic acid concentrations (2.8–100 μM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC‐analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]‐labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport‐assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high‐affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54‐1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 310–316, 1998.