Applied Microbiology and Biotechnology

, Volume 102, Issue 12, pp 5089–5103 | Cite as

Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding

Biotechnological products and process engineering
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Abstract

To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6–WS11, respectively. The strains WS3 (none) and WS4 (ΔnprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3–WS11, the pullulanase production levels of the resulting strains (WS3PUL–WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148.2 U/mL). Then, the scale-up pullulanase production levels of four recombinant strains WS5PUL, WS9PUL, WS10PUL, and WS11PUL were investigated in the 3-L fermenter cultivations. Strain WS9PUL produced the highest pullulanase activity (2449.6 U/mL) when fed an inorganic nitrogen source. However, the specific activity of the pullulanase obtained in a 3-L fermenter generally decreased as the number of protease deletions increased. Meanwhile, using pullulanase, α-cyclodextrin glucosyltransferase and β-cyclodextrin glucosyltransferase as reporter proteins, the protein production differences among strains WS3, WS9, and the widely used WB600 were investigated. Finally, the carbon to organic nitrogen source ratio of the feeding solution used in the 3-L fermenter was optimized. Recombinant strain WS9PUL fed with carbon and organic nitrogen sources in a ratio of 4:1 achieved a pullulanase activity of 5951.8 U/mL, the highest activity reported to date.

Keywords

Pullulanase Bacillus subtilis Protease Production Feeding solution 

Notes

Funding information

This work was funded by grants from the National Science Fund for Distinguished Young Scholars (31425020), the National Natural Science Foundation of China (31501419), the 111 Project (No. 111-2-06), and the Postgraduate Research & Practice Innovation Program of Jiangsu Provence (KYCX17_1416).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8965_MOESM1_ESM.pdf (495 kb)
ESM 1 (PDF 495 kb).

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxiChina
  3. 3.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina

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