Skip to main content
SpringerLink
Log in

In vivo functional expression of an extracellular Ca2+-independent Bacillus pumilus lipase in Bacillus subtilis WB800N

  • Original Article
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

A strain showing distinct lipase activity was isolated from food factory sewage and identified as Bacillus pumilus (named Bacillus pumilus Nws-bp1) by 16S rRNA sequence analysis. The wild-strain Nws-bp1 showed maximum lipase activity of 2.91 U/ml. Meanwhile, the lipase gene (named lip BP) was obtained from strain Nws-bp1 with the assistance of homology analysis. The gene has an open reading frame of 648 bp encoding 215-amino-acid lipase (LipBP) with 34-amino-acid putative signal peptide, and shows highest identity with the lipase from Bacillus pumilus MTCC B6033 (CP007436.1). Also, the lip BP gene without signal peptide sequence was expressed in Bacillus subtilis WB800N using amyQ (encoding an amylase) signal peptide. The lipase total enzyme activity was 44.15 U/ml which was about 15 times higher than that of the parent strain, and in supernatant was 32.29 U/ml (about 73 % of the total activity). The pH and temperature optima were pH 10.0 and 40 °C, respectively. Moreover, the recombinant LipBP showed apparent stability under alkaline conditions especially at pH 9.0–11.0. Also, LipBP showed stability under normal temperature and retained 85 % of the residual activity after incubation at 40 °C for 8 h without substrate. The specific activity of purified LipBP was 2650 ± 117 U/mg (pNPP substrate). The K m and V max values of purified LipBP were 1.36 mM and 208.25 μmol/(ml·min), respectively. This is the first report of Bacillus pumilus lipase expressed in Bacillus subtilis using amyQ signal peptide, and the pH stability and organic solvent tolerance recombinant lipase provide its potential value in industrial applications.

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.

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

Similar content being viewed by others

References

  • Cho AR, Yoo SK, Kim EJ (2000) Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 186:235–238

  • Dutta S, Ray L (2009) Production and characterization of an alkaline thermostable crude lipase from an isolated strain of Bacillus cereus C7. Appl Biochem Biotech 159:142–154

    Article  CAS  Google Scholar 

  • Ghanem EH, Al-Sayed HA, Saleh KM (2000) An alkalophilic thermostable lipase produced by a new isolate of Bacillus alcalophilus. World J Microbiol Biotechnol 16:459–464

    Article  CAS  Google Scholar 

  • Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64:763–781

    Article  CAS  PubMed  Google Scholar 

  • Ismael BJ, Yareth GT, Hugo CSU, Carmina M (2013) Immobilization and enantioselectivity of Bacillus pumilus lipase in ionic liquids. Mol Catal B 89:137–141

    Article  Google Scholar 

  • Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351

    Article  CAS  PubMed  Google Scholar 

  • Jose J, Kurup GM (1999) Purification and characterization of an extracellular lipase from a newly isolated thermophilic Bacillus pumilus. Indian J Exp Biol 37:1213–1217

    CAS  Google Scholar 

  • Kamijo T, Saito A, Ema S, Yoh I, Hayashi H, Nagata R, Nagata Y, Ando A (2011) Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01. Antonie van Leeuwenhoek 99:179–187

    Article  CAS  PubMed  Google Scholar 

  • Kim MH, Kim HK, Lee JK, Park SY, Oh TK (2000) Thermostable lipase of Bacillus stearothermophilus: high-level production, purification, and calcium-dependent thermostability. Biosci Biotech Biochem 64:280–286

  • Kim HK, Choi HJ, Kim MH, Sohn CB, Oh TK (2002) Expression and characterization of Ca2+-independent lipase from Bacillus pumilus B26. BBA Mol Cell Biol Lipids 1583:205–212

    Article  CAS  Google Scholar 

  • Kumar R, Mahajan S, Kumar A, Singh D (2011) Identification of variables and value optimization for optimum lipase production by Bacillus pumilus RK31 using statistical methodology. New Biotechnol 28:65–71

    Article  CAS  Google Scholar 

  • Lesuisse E, Schanck K, Colson C (1993) Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur J Biochem 216:155–160

    Article  CAS  PubMed  Google Scholar 

  • Lima VM, Krieger N, Mitchell DA, Baratti JC, Filippis ID, Fontana JD (2004) Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium. Mol Catal B 31:53–61

    Article  CAS  Google Scholar 

  • Liu ZQ, Zheng XB, Zhang SP, Zheng YG (2012) Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters. Microbiol Res 167:452–460

    Article  CAS  PubMed  Google Scholar 

  • Ma J, Zhang Z, Wang B, Kong X, Wang Y, Cao S, Feng Y (2006) Overexpression and characterization of a lipase from Bacillus subtilis. Protein Expr Purif 45:22–29

    Article  CAS  PubMed  Google Scholar 

  • Massadeh MI, Sabra FM (2013) Production and characterization of lipase from Bacillus stearothermophilus. Afr J Biotechnol 10(61):13139–13146

    Google Scholar 

  • Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51:711–729

    Article  CAS  PubMed  Google Scholar 

  • Pahoja VM, Sethar MA (2002) A review of enzymatic properties of lipase in plants, animals and microorganisms. Pak J Appl Sci 2:474–484

    Article  Google Scholar 

  • Pennisi E (1997) Biotechnology: in industry, extremophiles begin to make their mark. Science 276:705–706

    Article  CAS  PubMed  Google Scholar 

  • Phan TT, Nguyen HD, Schumann W (2006) Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif 46(2):189–195

    Article  CAS  PubMed  Google Scholar 

  • Rahman R, Chin J, Salleh A, Basri M (2003) Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y. Mol Genet Genomics 269:252–260

    CAS  PubMed  Google Scholar 

  • Rakesh K, Arpit S, Arun K, Deepak S (2012) Lipase from Bacillus pumilus RK31: production, purification and some properties. World Appl Sci J 16(7):940–948

    Google Scholar 

  • Rhee JK, Ahn DG, Kim YG, Oh JW (2005) New thermophilic and thermostable esterase with sequence similarity tothe hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol 71(2):817–825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17

    Article  CAS  PubMed  Google Scholar 

  • Schmid RD, Verger R (1998) Lipases: interfacial enzymes with attractive applications. Angew Chem Int Ed 37:1608–1633

    Article  Google Scholar 

  • Schmidt-Dannert C, Rúa ML, Atomi H, Schmid RD (1996) Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties. Biochim Biophys Acta 1301:105–114

    Article  PubMed  Google Scholar 

  • Shokri MM, Ahmadian S, Akbari N, Khajeh K (2014) Hydrophobic substitution of surface residues affects lipase stability in organic solvents. Mol Biotechnol 56(4):360–368

    Article  Google Scholar 

  • Sugihara A, Tani T, Tominaga Y (1991) Purification and characterization of a novel thermostable lipase from Bacillus sp. J Biochem 109:211–216

    CAS  PubMed  Google Scholar 

  • Tosato V, Bruschi CV (2004) Knowledge of the Bacillus subtilis genome: impacts on fundamental science and biotechnology. Appl Microbiol Biotechnol 64:1–6

    Article  CAS  PubMed  Google Scholar 

  • Wei W, Ma J, Guo S, Wei DZ (2014) A type I pullulanase of Bacillus cereus Nws-bc5 screening from stinky tofu brine: functional expression in Escherichia coli and Bacillus subtilis and enzyme characterization. Process Biochem 49:1893–1902

    Article  CAS  Google Scholar 

  • Westers H, Braun PG, Westers L, Antelmann H, Hecker M, Jongbloed JD, Yoshikawa H, Tanaka T, van Dijl JM, Quax WJ (2005) Genes involved in SkfA killing factor production protect a Bacillus subtilis lipase against proteolysis. Appl Environ Microbiol 71(4):1899–1908

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhang H, Zhang F, Li Z (2009) Gene analysis, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa. World J Microbiol Biotechnol 25:1267–1274

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was financially supported by the Fundamental Research Funds for the Central Universities of China, the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, the National Natural Science Foundation of China (No. C050203-31200596), the National High Technology Research and Development Program of China (No. 2013AA102109) and the National major science and technology projects of China (No. 2012ZX09304009).

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, People’s Republic of China

    Jing Ma, Yushu Ma, Wei Wei & Dong-zhi Wei

Authors
  1. Jing Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yushu Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dong-zhi Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Wei.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 321 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ma, J., Ma, Y., Wei, W. et al. In vivo functional expression of an extracellular Ca2+-independent Bacillus pumilus lipase in Bacillus subtilis WB800N. Ann Microbiol 65, 1973–1983 (2015). https://doi.org/10.1007/s13213-015-1035-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-015-1035-z

Keywords

Search

Navigation