Fermentation optimization, purification and biochemical characterization of ι-carrageenase from marine bacterium Cellulophaga baltica

Abstract

The ι-carrageenan degrading marine bacterium, Cellulophaga baltica, was isolated from the surface of a filamentous red alga Vertebrata fucoides. Maximum ι-carrageenase production was optimized by single-factor experiments. Optimal fermentation conditions were 1.6 g/L furcellaran, 4 g/L yeast extract as carbon sources, 5 g/L sea salt, and 48 h of incubation time at 20 °C. Extracellular ι-carrageenase from the culture supernatant was purified by ultrafiltration, ammonium sulfate precipitation, and finally by anion-exchange chromatography, showed a 26-fold increase in specific activity as compared to that in the crude enzyme. According to the results from SDS-PAGE and HPLC-SEC, the molecular weight of the purified enzyme was estimated to be 31 kDa. The purified enzyme showed the maximum specific activity of 571 U/mg at 40 °C and pH 7.5-8.0. It maintained 73% of the total activity below 40 °C and 90% of its total activity at pH 7.2. Notably, the enzyme is a cold-adapted ι-carrageenase, which showed 33.4% of the maximum activity at 10 °C. The enzyme was stimulated by Na⁺, K⁺, and NH₄⁺, whereas Ca²⁺, Mg²⁺, Fe³⁺, sea salt, and EDTA acted as enzyme inhibitors.

Keywords: Biochemical characterization; Cellulophaga baltica; ι-Carrageenase.