Stress fermentation strategies for the production of hyperthermostable superoxide dismutase from Thermus thermophilus HB27: effects of ions

Abstract

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 ${\text{NH}}_{ 4}^{{^{ + } }}$ and Mn²⁺ feeding. Under the optimized feeding time and concentrations of ${\text{NH}}_{ 4}^{{^{ + } }}$ 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 ${\text{NH}}_{ 4}^{{^{ + } }}$ 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 ${\text{NH}}_{ 4}^{{^{ + } }}$ 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 ${\text{NH}}_{ 4}^{{^{ + } }}$ 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.