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

  • Kang Zhang
  • Lingqia Su
  • Jing Wu
Biotechnological products and process engineering


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.


Pullulanase Bacillus subtilis Protease Production Feeding solution 


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).


  1. Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746PubMedPubMedCentralGoogle Scholar
  2. Chen WB, Nie Y, Xu Y (2013) Signal peptide-independent secretory expression and characterization of pullulanase from a newly isolated Klebsiella variicola SHN-1 in Escherichia coli. Appl Biochem Biotechnol 169:41–54CrossRefPubMedGoogle Scholar
  3. Chen A, Li Y, Liu X, Long Q, Yang Y, Bai Z (2014) Soluble expression of pullulanase from Bacillus acidopullulyticus in Escherichia coli by tightly controlling basal expression. J Ind Microbiol Biotechnol 41:1803–1810CrossRefPubMedGoogle Scholar
  4. Coutte F, Leclere V, Bechet M, Guez JS, Lecouturier D, Chollet-Imbert M, Dhulster P, Jacques P (2010) Effect of pps disruption and constitutive expression of srfA on surfactin productivity, spreading and antagonistic properties of Bacillus subtilis 168 derivatives. J Appl Microbiol 109:480–491PubMedGoogle Scholar
  5. Dempsey LA, Dubnau DA (1989) Localization of the replication origin of plasmid pE194. J Bacteriol 171:2866–2869CrossRefPubMedPubMedCentralGoogle Scholar
  6. Duan X, Chen J, Wu J (2013) Optimization of pullulanase production in Escherichia coli by regulation of process conditions and supplement with natural osmolytes. Bioresour Technol 146:379–385CrossRefPubMedGoogle Scholar
  7. Gupta M, Rao KK (2014) Phosphorylation of DegU is essential for activation of amyE expression in Bacillus subtilis. J Biosci 39:747–752CrossRefPubMedGoogle Scholar
  8. Gupta R, Beg QK, Khan S, Chauhan B (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl Microbiol Biotechnol 60:381–395CrossRefPubMedGoogle Scholar
  9. Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: an unfolding story. Trends Microbiol 16:73–79CrossRefPubMedGoogle Scholar
  10. Harwood CR, Wipat A (1996) Sequencing and functional analysis of the genome of Bacillus subtilis strain 168. FEBS Lett 389:84–87CrossRefPubMedGoogle Scholar
  11. Hii SL, Tan JS, Ling TC, Ariff AB (2012) Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res 2012:921362CrossRefPubMedPubMedCentralGoogle Scholar
  12. Huang J, Du Y, Xu G, Zhang H, Zhu F, Huang L, Xu Z (2011) High yield and cost-effective production of poly(gamma-glutamic acid) with Bacillus subtilis. Eng Life Sci 11:291–297CrossRefGoogle Scholar
  13. Kabisch J, Thuermer A, Huebel T, Popper L, Daniel R, Schweder T (2013) Characterization and optimization of Bacillus subtilis ATCC 6051 as an expression host. J Biotechnol 163:97–104CrossRefPubMedGoogle Scholar
  14. Kelly RM, Dijkhuizen L, Leemhuis H (2009) The evolution of cyclodextrin glucanotransferase product specificity. Appl Microbiol Biotechnol 84:119–133CrossRefPubMedPubMedCentralGoogle Scholar
  15. Krishnappa L, Dreisbach A, Otto A, Goosens VJ, Cranenburgh RM, Harwood CR, Becher D, van Dijl JM (2013) Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis. J Proteome Res 12:4101–4110CrossRefPubMedGoogle Scholar
  16. Krishnappa L, Monteferrante CG, Neef J, Dreisbach A, van Dijl JM (2014) Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis. Appl Environ Microbiol 80:1463–1468CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kuo CC, Lin CA, Chen JY, Lin MT, Duan KJ (2009) Production of cyclodextrin glucanotransferase from an alkalophilic Bacillus sp. by pH-stat fed-batch fermentation. Biotechnol Lett 31:1723–1727CrossRefPubMedGoogle Scholar
  18. Kwon EY, Kim KM, Kim MK, Lee IY, Kim BS (2011) Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis. Bioprocess Biosyst Eng 34:789–793CrossRefPubMedGoogle Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lejeune A, Sakaguchi K, Imanaka T (1989) A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (alpha-cyclodextrin) glucanotransferase. Anal Biochem 181:6–11CrossRefPubMedGoogle Scholar
  21. Li WF, Zhou XX, Lu P (2004) Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol 155:605–610CrossRefPubMedGoogle Scholar
  22. Margot P, Karamata D (1996) The wprA gene of Bacillus subtilis 168, expressed during exponential growth, encodes a cell-wall-associated protease. Microbiology 142:3437–3444CrossRefPubMedGoogle Scholar
  23. Moller MS, Henriksen A, Svensson B (2016) Structure and function of alpha-glucan debranching enzymes. Cell Mol Life Sci 73:2619–2641CrossRefPubMedGoogle Scholar
  24. Nguyen TT, Quyen TD, Le HT (2013) Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microb Cell Factories 12:79CrossRefGoogle Scholar
  25. Penninga D, vanderVeen BA, Knegtel RMA, vanHijum S, Rozeboom HJ, Kalk KH, Dijkstra BW, Dijkhuizen L (1996) The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J Biol Chem 271:32777–32784CrossRefPubMedGoogle Scholar
  26. Pohl S, Harwood CR (2010) Heterologous protein secretion by Bacillus species from the cradle to the grave. Adv Appl Microbiol 73:1–25CrossRefPubMedGoogle Scholar
  27. Pohl S, Bhavsar G, Hulme J, Bloor AE, Misirli G, Leckenby MW, Radford DS, Smith W, Wipat A, Williamson ED, Harwood CR, Cranenburgh RM (2013) Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins. Proteomics 13:3298–3308CrossRefPubMedGoogle Scholar
  28. Shankar R, Madihah MS, Shaza EM, Aswati NKO, Suraini AA, Kamarulzaman K (2014) Application of different feeding strategies in fed batch culture for pullulanase production using sago starch. Carbohydr Polym 102:962–969CrossRefGoogle Scholar
  29. Shi M, Chen Y, Yu S, Gao Q (2013) Preparation and properties of RS III from waxy maize starch with pullulanase. Food Hydrocolloid 33:19–25CrossRefGoogle Scholar
  30. Stephenson K, Harwood CR (1998a) Influence of a cell-wall-associated protease on production of alpha-amylase by Bacillus subtilis. Appl Environ Microbiol 64:2875–2881PubMedPubMedCentralGoogle Scholar
  31. Stephenson K, Harwood CR (1998b) Influence of a cell-wall-associated protease on production of alpha-amylase by Bacillus subtilis. Appl Environ Microbiol 64:2875–2881PubMedPubMedCentralGoogle Scholar
  32. Stephenson K, Bron S, Harwood CR (1999) Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies. Lett Appl Microbiol 29:141–145CrossRefGoogle Scholar
  33. Tjalsma H, Antelmann H, Jongbloed JD, Braun PG, Darmon E, Dorenbos R, Dubois JY, Westers H, Zanen G, Quax WJ, Kuipers OP, Bron S, Hecker M, van Dijl JM (2004) Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol Mol Biol Rev 68:207–233CrossRefPubMedPubMedCentralGoogle Scholar
  34. van der Veen BA, Uitdehaag JC, Dijkstra BW, Dijkhuizen L (2000) Engineering of cyclodextrin glycosyltransferase reaction and product specificity. Biochim Biophys Acta 1543:336–360CrossRefPubMedGoogle Scholar
  35. Veening JW, Igoshin OA, Eijlander RT, Nijland R, Hamoen LW, Kuipers OP (2008) Transient heterogeneity in extracellular protease production by Bacillus subtilis. Mol Syst Biol 4:184CrossRefPubMedPubMedCentralGoogle Scholar
  36. Waldeck J, Meyer-Rammes H, Wieland S, Feesche J, Maurer KH, Meinhardt F (2007) Targeted deletion of genes encoding extracellular enzymes in Bacillus licheniformis and the impact on the secretion capability. J Biotechnol 130:124–132CrossRefPubMedGoogle Scholar
  37. Wenzel M, Mueller A, Siemann-Herzberg M, Altenbuchner J (2011) Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation. Appl Environ Microbiol 77:6419–6425CrossRefPubMedPubMedCentralGoogle Scholar
  38. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694:299–310CrossRefPubMedGoogle Scholar
  39. Westers H, Westers L, Darmon E, van Dijl JM, Quax WJ, Zanen G (2006) The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis. FEBS J 273:3816–3827CrossRefPubMedGoogle Scholar
  40. Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173:4952–4958CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wu SC, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68:3261–3269CrossRefPubMedPubMedCentralGoogle Scholar
  42. Wu Q-L, Chen T, Gan Y, Chen X, Zhao X-M (2007) Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Appl Microbiol Biotechnol 76:783–794CrossRefPubMedGoogle Scholar
  43. Wu J, Zhang K, Su L, Huang Y (2017) A Bacillus subtilis strain for efficient heterologous protein expression and high-density cultivation. China Patent CN106754466AGoogle Scholar
  44. Xu B, Yang Y-J, Huang Z-X (2006) Cloning and overexpression of gene encoding the pullulanase from Bacillus naganoensis in Pichia pastoris. J Ind Microbiol Biotechnol 16:1185–1191Google Scholar
  45. Zhang K, Duan X, Wu J (2016) Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci Rep 6:strep27943CrossRefGoogle Scholar
  46. Zhang K, Su L, Duan X, Liu L, Wu J (2017) High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system. Microb Cell Factories 16:32CrossRefGoogle Scholar
  47. Zou C, Duan X, Wu J (2014) Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy. Bioresour Technol 172:174–179CrossRefPubMedGoogle Scholar
  48. Zou C, Duan X, Wu J (2016a) Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis. J Ind Microbiol Biotechnol 43:495–504CrossRefPubMedGoogle Scholar
  49. Zou C, Duan X, Wu J (2016b) Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis. Appl Microbiol Biotechnol 100:7115–7123CrossRefPubMedGoogle Scholar

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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