Applied Microbiology and Biotechnology

, Volume 100, Issue 16, pp 7115–7123 | Cite as

Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis

  • Chun Zou
  • Xuguo Duan
  • Jing Wu
Biotechnologically relevant enzymes and proteins


Addition of MgCl2 to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis. The specific activity of the pullulanase obtained from medium supplemented with MgCl2 was also higher than that obtained in culture medium without added magnesium ions. In this work, the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Circular dichroism assays revealed considerable differences in secondary structure between the active and inactive pullulanase forms. Transmission electron microscopy suggested that magnesium ion addition inhibits the shedding of cell wall protein (HWP) layers from the cell surface. Quantitative real-time PCR showed that magnesium ion addition represses transcription of HWP. Because the pullulanase gene and HWP have identical promoters, pullulanase gene transcription was also inhibited. These results suggest that when pullulanase is expressed slowly, it tends to fold into an active form.


Pullulanase Brevibacillus choshinensis Magnesium ions Active form Cell wall protein 



This work received financial support from the National Science Foundation for Distinguished Young Scholars (31425020), the National Natural Science Foundation of China (31271813, 31401636), the Project of Outstanding Scientific and Technological Innovation Group of Jiangsu Province, the Natural Science Foundation of Jiangsu Province (BK20140142), and the 111 Project (No. 111-2-06).

Compliance with ethical standards

Conflict of interest

The authors confirm that they have no conflicts of interests.

Ethical approval

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

Supplementary material

253_2016_7386_MOESM1_ESM.pdf (265 kb)
ESM 1 (PDF 264 kb)


  1. Adachi T, Yamagata H, Tsukagoshi N, Udaka S (1991) Repression of the cell wall protein gene operon in Bacillus brevis 47 by magnesium and calcium ions. J Bacteriol 173:4243–4245PubMedPubMedCentralGoogle Scholar
  2. Blanco CA, Caballero I, Barrios R, Rojas A (2014) Innovations in the brewing industry: light beer. Int J Food Sci Nutr 65(6):655–660CrossRefPubMedGoogle Scholar
  3. Chen A, Li YM, Liu XX, Long Q, Yang YK, Bai ZH (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. Cheng S (2015) Expression, thermostability modification and application of sucrose isomerase from Serratia plymuthica [D]. Jiangnan UniversityGoogle Scholar
  5. Duan X, Wu J (2015) Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation. Appl Environ Microb 81:1926–1931CrossRefGoogle 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. Duan X, Zou C, Wu J (2015) Triton X-100 enhances the solubility and secretion ratio of aggregation-prone pullulanase produced in Escherichia coli. Bioresour Technol 194:137–143CrossRefPubMedGoogle Scholar
  8. Ebisu S, Tsuboi A, Takagi H, Naruse Y, Yamagata H, Tsukagoshi N, Udaka S (1990) Conserved structures of cell-wall protein genes among protein-producing Bacillus-brevis strains. J Bacteriol 172:1312–1320PubMedPubMedCentralGoogle Scholar
  9. Gohel V, Duan G (2012) Conventional process for ethanol production from Indian broken rice and pearl millet. Bioproc Biosyst Eng 35:1297–1308CrossRefGoogle Scholar
  10. Hii SL, Tan JS, Ling TC, Ariff AB (2012) Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res 2012:921362CrossRefPubMedPubMedCentralGoogle Scholar
  11. Li ZF, Li B, Liu ZG, Wang M, Gu ZB, Du GC, Wu J, Chen J (2009) Calcium leads to further increase in glycine-enhanced extracellular secretion of recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli. J Agric Food Chem 57:6231–6237CrossRefPubMedGoogle Scholar
  12. Li S, Xu H, Yu J, Wang Y, Feng X, Ouyang P (2013) Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization. Bioproc Biosyst Eng 36:1395–1405CrossRefGoogle Scholar
  13. Lin QL, Xiao HX, Liu GQ, Liu ZH, Li LH, Yu FX (2013) Production of maltose syrup by enzymatic conversion of rice starch. Food Bioprocess Tech 6:242–248CrossRefGoogle Scholar
  14. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4):402–408CrossRefPubMedGoogle Scholar
  15. Mizukami M, Hanagata H, Miyauchi A (2010) Brevibacillus expression system: host-vector system for efficient production of secretory proteins. Curr Pharm Biotechnol 11:251–258CrossRefPubMedGoogle Scholar
  16. Mizukami M, Tokunaga H, Onishi H, Ueno Y, Hanagata H, Miyazaki N, Kiyose N, Ito Y, Ishibashi M, Hagihara Y, Arakawa T, Miyauchi A, Tokunaga M (2014) Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system. Protein Expr Purif 105:23–32CrossRefPubMedGoogle Scholar
  17. Mu T, Liang W, Ju Y, Wang Z, Wang Z, Roycik MD, Sang QX, Yu D, Xiang H, Fang X (2013) Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis. Protein Expr Purif 91:125–133CrossRefPubMedGoogle Scholar
  18. Sidhu MS, Olsen I (1997) S-layers of Bacillus species. Microbiol-Sgm 143:1039–1052CrossRefGoogle Scholar
  19. Singh RS, Saini GK, Kennedy JF (2010) Maltotriose syrup preparation from pullulan using pullulanase. Carbohydr Polym 80(2):401–407CrossRefGoogle Scholar
  20. Sreerama N, Woody RW (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal Biochem 209(1):32–44CrossRefPubMedGoogle Scholar
  21. Talekar S, Pandharbale A, Ladole M, Nadar S, Mulla M, Japhalekar K, Arage D (2013) Carrier free co-immobilization of alpha-amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity. Bioresour Technol 147:269–275CrossRefPubMedGoogle Scholar
  22. Udaka S, Yamagata H (1993) Protein secretion in Bacillus brevis. Antonie Van Leeuwenhoek 64:137–143CrossRefPubMedGoogle Scholar
  23. Udaka S, Tsukagoshi N, Yamagata H (1989) Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol Genet Eng Rev 7:113–146CrossRefPubMedGoogle Scholar
  24. Van Mellaert L, Anné J (2002) Gram-positive bacteria as host cells for heterologous production of biopharmaceuticals[M]//novel frontiers in the production of compounds for biomedical use. Springer, Netherlands, pp. 277–300Google Scholar
  25. Wu J, Duan X, Fang B (2015) A strain for α-cyclodextrin glycosyltransferase production and its application. China Patent CN201510056200.3Google Scholar
  26. Yamada H, Tsukagoshi N, Udaka S (1981) Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47. J Bacteriol 148:322–332PubMedPubMedCentralGoogle Scholar
  27. Zhang HX, Jin ZY (2011) Preparation of resistant starch by hydrolysis of maize starch with pullulanase. Carbohydr Polym 83:865–867CrossRefGoogle Scholar
  28. Zhang H, Tian Y, Bai Y, Xu X, Jin Z (2013) Structure and properties of maize starch processed with a combination of α-amylase and pullulanase. Int J Biol Macromol 52:38–44CrossRefPubMedGoogle Scholar
  29. 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
  30. Zou C, Duan XG, Wu J (2015) Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis. J Ind Microbiol Biotechnol 1-10. doi: 10.1007/s10295-015-1719-1

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© Springer-Verlag Berlin Heidelberg 2016

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

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