期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 159, 期 -, 页码 42-48出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2019.03.006
关键词
Pullulanase; Bacillus subtilis; Fermentation optimization; Integrative expression; Homologous recombination
类别
资金
- National Natural Science Foundation of China (NSFC) [31872891, 21676120]
- Program for Advanced Talents within Six Industries of Jiangsu Province [2015-NY-007]
- 111 Project [111-2-06]
- High-end Foreign Experts Recruitment Program [GDT20183200136]
- National Program for Support of Top-notch Young Professionals
- Fundamental Research Funds for the Central Universities [JUSRP51504]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Top-notch Academic Programs Project of Jiangsu Higher Education Institutions
- Jiangsu province Collaborative Innovation Center for Advanced Industrial Fermentation industry development program
Pullulanase is widely used in the starch processing industry as a debranching enzyme. However, extracellular production of pullulanase from recombinant Bacillus subtilis is limited and the loss of plasmids during fermentation of B. subtilis recombinants seriously affects the expression of the foreign protein, especially in large-scale production. In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pill gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, P-H(pall), and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 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-P-Hpall and B. subtilis WB600-P-Hpall-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 onefactor-at-a-time technique for B. subtilis WB800-P(Hupall)pul. Consequently, the extracellular production of PUL was significantly enhanced, resulting in an activity of 60.85 U/ml.
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