Psuedoalteromonas carrageenovora로부터 k-카라기난 분해효소 유전자 cloning과 재조합 단백질 대량 생산

Abstract

The cgkA gene of Pseudoalteromonas carrageenovora encoding a κ-carrageenase was cloned by PCR. The recombinat plasmid, pKC44a, was constructed by ligation of PCR product in pET44a vector system. pKC44a transformed into E. coli BL21(DE3) was incubated in LB broth at 37℃ for 2 hr 20 min. Recombinant κ-carrageenase was expressed by IPTG, to give final concentration of 0.1 mM at 20℃. The recombinant enzyme was excreted in the enzyme culture medium and the activity was increased as time progressed. The protien expressed from pKC44a was purified by ionic exchange chromatographied. The molecular weight of recombinant κ-carrageenase was estimated as 42 kDa by SDS-PAGE. The purified κ-carrageenase had maximal activity at 35℃ and pH 8.0 for 4 hrs.