Electroporation and Recombinant Strain Screening

The recombinant plasmids pPIC9K-cgkZ, pPIC9K-cgkZΔPst and pPIC9K-cgkZΔCBM were linearized using SalI and transformed into P. pastoris by electroporation using a MicroPulser^TM electroporator (Bio-Rad, Hercules, CA, United States) at a voltage of 2 kV. After electroporation, the yeast cells were cultured in MD medium at 30°C for 3 days. Then the yeast cells were further diluted with sterile water and cultured on a YPD agar plate containing 4 mg/mL of geneticin (G418) at 30°C for another 3 days. The obtained colonies were activated in 10 mL of YPD medium at 28°C for 24 h, and then 2 mL of medium was transferred to a 1 L shaker flask containing 200 mL BMGY medium. P. pastoris was cultured at 28°C for 72 h with the addition of methanol (1%, v/v) every 24 h. After incubation, the culture was centrifuged and the supernatant was retained to measure κ-carrageenase activity. Enzymatic activity was measured as described by Liu et al. (2013) and protein concentration was measured using the Bradford method (Liu et al., 2011). Strains exhibiting high enzymatic activity were screened and stored at -80°C.