Abstract:The effects of agitation on fragmentation of a recombinant strain of Aspergillus oryzae and its consequential effects on protein production have been investigated. Constant mass, 5.3-L chemostat cultures at a dilution rate of 0.05 h −1 and a dissolved oxygen level of 75% air saturation, have been conducted at 550, 700, and 1000 rpm. These agitation speeds were chosen to cover a range of specific power inputs (2.2 to 12 kW m −3 ) from realistic industrial levels to much higher values. The use of a constant mass chemostat linked to a gas blender allowed variation of agitation speed and hence gas holdup without affecting the dilution rate or the concentration of dissolved oxygen. The morphology of both the freely dispersed mycelia and clumps was characterized using image analysis. Statistical analysis showed that it was possible to obtain steady states with respect to morphology. The mean projected area at each steady state under growing conditions correlated well with the ''energy dissipation/circulation'' function, [P/(kD 3 t c )], where P is the power input, D the impeller diameter, t c the mean circulation time, and k is a geometric constant for a given impeller. Rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation. Protein production (␣-amylase and amyloglucosidase) was found to be independent of agitation speed in the range 550 to 1000 rpm (P/V = 2.2 and 12.6 kW m −3 , respectively), although significant changes in mycelial morphology could be measured for similar changes in agitation conditions. This suggests that mycelial morphology does not directly affect protein production (at a constant dilution rate and, therefore, specific growth rate). An understanding of how agitation affects mycelial morphology and productivity would be valuable in optimizing the design and operation of large-scale fungal fermentations for the production of recombinant proteins.

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 ؎ 89 ng g ؊1 fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 ؎ 309 ng g ؊1 fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g ؊1 fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35°C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5,5-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K m of 65 M and a V max of 83 nmol min ؊1 mg ؊1 protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.

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