Enhancement of the production of Bacillus naganoensis pullulanase in recombinant Bacillus subtilis by integrative expression

In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pul gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, PHpaII, and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 and B. subtilis WB600 by double homologous recombination. The pullulanase activity of up to 30.32 U/ml and 18.83 U/ml was achieved for B. subtilis WB800-PHpaII-pul and B. subtilis WB600-PHpaII-pul, respectively, under primary conditions. To further enhance the yield of PUL, the effects of four important factors (inoculum size, incubation temperature, shaking speed, and initial pH) on the expression of PUL in shake flask fermentation were evaluated by “one-factor-at-a-time” technique for B. subtilis WB800-PHpaII-pul. Consequently, the extracellular production of PUL was significantly enhanced, resulting in an activity of 60.85 U/ml.

Source: Protein Expression and Purification - March 18, 2019 Category: Biochemistry Source Type: research