Skip to main content
SpringerLink
Log in

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

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • 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 173:4243–4245

    CAS  PubMed  PubMed Central  Google Scholar 

  • Blanco CA, Caballero I, Barrios R, Rojas A (2014) Innovations in the brewing industry: light beer. Int J Food Sci Nutr 65(6):655–660

    Article  CAS  PubMed  Google Scholar 

  • 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 41:1803–1810

    Article  CAS  PubMed  Google Scholar 

  • Cheng S (2015) Expression, thermostability modification and application of sucrose isomerase from Serratia plymuthica [D]. Jiangnan University

  • 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 81:1926–1931

    Article  CAS  Google Scholar 

  • 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 146:379–385

    Article  CAS  PubMed  Google Scholar 

  • 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 194:137–143

    Article  CAS  PubMed  Google Scholar 

  • 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 172:1312–1320

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gohel V, Duan G (2012) Conventional process for ethanol production from Indian broken rice and pearl millet. Bioproc Biosyst Eng 35:1297–1308

    Article  CAS  Google Scholar 

  • Hii SL, Tan JS, Ling TC, Ariff AB (2012) Pullulanase: role in starch hydrolysis and potential industrial applications. Enzyme Res 2012:921362

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 57:6231–6237

    Article  CAS  PubMed  Google Scholar 

  • 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 36:1395–1405

    Article  CAS  Google Scholar 

  • 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 6:242–248

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4):402–408

    Article  CAS  PubMed  Google Scholar 

  • Mizukami M, Hanagata H, Miyauchi A (2010) Brevibacillus expression system: host-vector system for efficient production of secretory proteins. Curr Pharm Biotechnol 11:251–258

    Article  CAS  PubMed  Google Scholar 

  • 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 105:23–32

    Article  PubMed  Google Scholar 

  • 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 91:125–133

    Article  CAS  PubMed  Google Scholar 

  • Sidhu MS, Olsen I (1997) S-layers of Bacillus species. Microbiol-Sgm 143:1039–1052

    Article  CAS  Google Scholar 

  • Singh RS, Saini GK, Kennedy JF (2010) Maltotriose syrup preparation from pullulan using pullulanase. Carbohydr Polym 80(2):401–407

    Article  CAS  Google Scholar 

  • Sreerama N, Woody RW (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal Biochem 209(1):32–44

    Article  CAS  PubMed  Google Scholar 

  • 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 147:269–275

    Article  CAS  PubMed  Google Scholar 

  • Udaka S, Yamagata H (1993) Protein secretion in Bacillus brevis. Antonie Van Leeuwenhoek 64:137–143

    Article  PubMed  Google Scholar 

  • Udaka S, Tsukagoshi N, Yamagata H (1989) Bacillus brevis, a host bacterium for efficient extracellular production of useful proteins. Biotechnol Genet Eng Rev 7:113–146

    Article  CAS  PubMed  Google Scholar 

  • 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. 277–300

    Google Scholar 

  • Wu J, Duan X, Fang B (2015) A strain for α-cyclodextrin glycosyltransferase production and its application. China Patent CN201510056200.3

  • 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 148:322–332

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang HX, Jin ZY (2011) Preparation of resistant starch by hydrolysis of maize starch with pullulanase. Carbohydr Polym 83:865–867

    Article  CAS  Google Scholar 

  • 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 52:38–44

    Article  PubMed  Google Scholar 

  • 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 172:174–179

    Article  CAS  PubMed  Google Scholar 

  • 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

Download references

Acknowledgments

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).

Author information

Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Chun Zou, Xuguo Duan & Jing Wu

  2. School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Chun Zou, Xuguo Duan & Jing Wu

Authors
  1. Chun Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xuguo Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jing Wu.

Ethics declarations

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.

Electronic supplementary material

ESM 1

(PDF 264 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zou, C., Duan, X. & Wu, J. Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis . Appl Microbiol Biotechnol 100, 7115–7123 (2016). https://doi.org/10.1007/s00253-016-7386-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7386-y

Keywords

Search

Navigation