The expression of oligo-1,6-glucosidase from Bacillus subtilis in Pichia pastoris

According to the published sequence of oligo-1,6-glucosidase gene from Bacillus subtilis, a pair of primers was designed to amplify the oligo-1,6glucosidase gene. The PCR fragment was treated by T_4 DNA polymerase and dTTP,then ligated with the expression vector pHBM905 which has digested by CpoI and NotI. A recombinant vector pHBM9053 was acquired and transformed into Pichia pastoris GS115, KM71, SMD1168 respectively. Oligo-1,6-glucosidase was expressed in all this three strains.The recombinant Pichia pastoris GS115 ((pHBM9053)) has the highest level of expression oligo-1,6-glucosidase of 2.223u/mL,compared with other two recombinant strains KM71 and SMD1168. The oligo-1,6glucosidase expressed by GS115( pHBM9053) has a pH optimum of 6 and a temperature optimum of 75℃, and are active at pH ranged from 8 to 11 and temperature ranged from 30℃ to 75℃.