Abstract: The pullulanase of Bacillus acidopullulyticus has low secretory expression level as a macromolecule. For pullulanase secretory in recombination Escherichia coli BL21(DE3), BL21(DE3)-pET-28a(+)-pelB-pul13A with PelB signal peptide was constructed, then fermentation optimization and medium additives were employed for better secretory. Through the construction of recombinant strain BL21(DE3) with pelB, pullulanase was secreted at a low level. By fermentation optimization including medium , induced temperature , the concentration of IPTG, induced time and medium additives, the extracellular pullulanase activity was markedly increased. pullulanase was secreted into periplasmic space. Under the condition of 0.1 mmol/L IPTG, 20℃ induced after D600 nm was about 5.0-6.0, the periplasmic pullulanase activity reached to 102.5 U/mL with addition of 0.03%SDS after induction for 20 h.

Key words: pullulanase, secretory expression, signal peptide, fermentation optimization, medium additives, SDS