Efficient production of a recombinant ι-carrageenase in Brevibacillus choshinensis using a new integrative vector for the preparation of ι-carrageenan …

In this study, a new integrative vector (pBCGA) was developed and used for the high-level expression of an optimized ι-carrageenase gene (CGIOP) in Brevibacilluschoshinensis. The pBCGA vector allowed multiple copies of the gene CGIOP to be stably integrated into the genomic DNA of B. choshinensis. The recombinant strain I24 could produce an extracellular ι-carrageenase activity of 38.9 U/mL within 72 h, which remained stable after five sequential inoculations and cultivations under the antibiotic-free culture conditions. Furthermore, the strain I24 was applied to 10-L fermentation under the antibiotic-free culture condition, resulting in the highest observed ι-carrageenase activity of 182.4 U/mL within 24 h. Subsequently, recombinant ι-carrageenase (rCgiA) was purified and characterized, exhibiting an optimal pH and temperature of 8.0 and 45 °C, respectively. Notably, rCgiA showed considerable stability below 45 °C and over a relatively broad pH range of 6.0–11.0. In addtion, the activity of rCgiA was significantly stimulated by NaCl, Mg2+, and Ca2+. HILIC LC-LTQ-Orbitrap-FTMS analysis revealed that rCgiA hydrolyzed ι-carrageenan via a processive mechanism with the major product of ι-carrageenan tetrasaccharide. Thus, the strain B. choshinensis I24 had broad potential for use in the environment-friendly and large-scale production of ι-carrageenase and ι-carrageenan oligosaccharides.