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In this study, we explored how ammonium and metal ion stresses affected the production of recombinant hyperthermostable manganese superoxide dismutase (Mn-SOD). To improve Mn-SOD production, fed-batch culture in shake flasks and bioreactor fermentation were undertaken to examine the effects of NH4+ and Mn2+ feeding. Under the optimized feeding time and concentrations of NH4+ and Mn2+, the maximal SOD activity obtained from bioreactor fermentation reached some 480 U/ml, over 4 times higher than that in batch cultivation (113 U/ml), indicating a major enhancement of the concentration of Mn-SOD in the scale-up of hyperthermostable Mn-SOD production. In contrast, when the fed-batch culture with appropriate NH4+ and Mn2+ feeding was carried out in the same 5-L stirred tank bioreactor, a maximal SOD concentration of some 450 U/ml was obtained, again indicating substantial increase in SOD activity as a result of NH4+ and Mn2+ feeding. The isoelectric point (pI) of the sample was found to be 6.2. It was highly stable at 90 °C and circular dichroism measurements indicated a high α-helical content of 70 % as well, consistent with known SOD properties. This study indicates that NH4+ and Mn2+ play important roles in Mn-SOD expression. Stress fermentation strategies established in this study are useful for large-scale efficient production of hyperthermostable Mn-SOD and may also be valuable for the scale-up of other extremozymes. © 2013 Springer Japan.

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995 - 1002