High expression of agarase AgaD in Escherichia coli

Abstract:

Abstract:［Objective］ We constructed highly efficient expression systems for agarase AgaD and optimized its culture conditions．［Methods］First，the codon usage of AgaD was optimized to make it suitable for expression in E.coli． Then，the gene expression vector was transformed into different E．coli hosts． According to the“N-end rule”that is related to the in vivo half-life of a protein，a mutant was constructed． Finally，the effects of CaCl2 and glycine on enzyme production were evaluated．［Results］A highly efficient expression system of agarase AgaD was constructed，named pET-22b(+)-optagaDx-AD494 (DE3)．Replacing N-terminal second amino acid phenylalanine with alanine significantly improved agarase production and shortened the fermentation period．The extracellular enzyme activity was further up-regulated by CaCl2 and glycine． After optimization，the extracellular enzyme production raised from 20 U/L to 11300 U/L，more than 500 folds.［Conclusion］The high expression system of AgaD provides good basis for further studying agarases.