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Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01

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Abstract

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl2, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.

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Abbreviations

CBB:

Coomassie Brilliant Blue

N-terminal:

Amino-terminal

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

References

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R (2007) Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25:1007–1014

    Article  CAS  PubMed  Google Scholar 

  • Cho A-R, Yoo S-K, Kim E-J (2000) Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 186:235–238

    Article  CAS  PubMed  Google Scholar 

  • Dartois V, Baulard A, Schanck K, Colson C (1992) Cloning nucleotide sequence and expression of in Escherichia coli of a lipase gene from Bacillus subtilis 168. Biochem Biophys Acta 1131:253–260

    CAS  PubMed  Google Scholar 

  • Eggert T, Pencreac’h G, Douchet I, Verger R, Jaeger K-E (2000) A novel extracellular esterase from Bacillus subtilis and its convertion to a monoacylglycerol hydrolase. Eur J Biochem 267:6459–6469

    Article  CAS  PubMed  Google Scholar 

  • Eggert T, van Pouderoyen G, Dijkstra BW, Jaeger K-E (2001) Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure. FEBS Lett 502:89–92

    Article  CAS  PubMed  Google Scholar 

  • Eggert T, Brockmeier U, Dröge MJ, Quax WJ, Jaeger K-E (2003) Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH. FEMS Microbiol Lett 225:319–324

    Article  CAS  PubMed  Google Scholar 

  • Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP, and related tools. Nat Protoc 2:953–971

    Article  CAS  PubMed  Google Scholar 

  • Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC

    Google Scholar 

  • Kim H-K, Park S-Y, Lee J-K, Oh T-K (1998) Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci Biotechnol Biochem 62:6–71

    Google Scholar 

  • Kimura K, Kubo M (2006) Fats and oil-containing waste water treatment with fats and oils-degrading microorganisms. Oleoscience 6:501–506 (in Japanese)

    Google Scholar 

  • Koma D, Hasumi F, Yamanoto E, Ohta T, Chung S-Y, Kubo M (2001) Biodegradation of long-chain n-paraffins from waste oil of Ca engine by Acinetobacter sp. J Biosci Bioeng 91:94–96

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lee D-W, Koh Y-S, Kim K-J, Kim B-C, Choi H-J, Kim D-S, Suhartono MT, Pyun Y-R (1999) Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 179:393–400

    Article  CAS  PubMed  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 

  • Nthangeni MB, Patterton H-G, van Tonder A, Vergeer WP, Litthauer D (2001) Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. Enzym Microb Technol 28:705–712

    Article  CAS  Google Scholar 

  • Patkar SA, Bjorkling F (1994) Lipase inhibitors. In: Woolley P, Peterson SB (eds) Lipases-their structure, biochemistry and application. Cambridge University Press, Cambridge, pp 207–224

    Google Scholar 

  • Ruiz C, Blanco A, Javier Pastor FI, Diaz P (2002) Analysis of Bacillus megaterium lipolytic system and cloning of LipA, a novel subfamily I.4 bacterial lipase. FEMS Microbiol Lett 217:263–267

    Article  CAS  PubMed  Google Scholar 

  • Ruiz C, Javier Pastor FI, Diaz P (2003) Isolation and characterization of Bacillus sp. BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species. Lett Appl Microbiol 37:354–359

    Article  CAS  PubMed  Google Scholar 

  • Saito A, Shinya T, Miyamoto K, Yokoyama T, Kaku H, Minami E, Shibuya N, Tsujibo H, Nagata Y, Ando A, Fujii T, Miyashita K (2007) The dasABC gene cluster, adjacent to dasR, encodes a novel ABC transporter for the uptake of N,N′-diacetylchitobiose in Streptomyces coelicolor A3(2). Appl Environ Microbiol 73:3000–3008

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sánchez M, Prim N, Rández-Gil F, Pastor FIJ, Diaz P (2002) Engineering of baker’s yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis lipase A. Biotechnol Bioeng 78:339–345

    Article  PubMed  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

    PubMed  Google Scholar 

  • Thanomsub BW, Boonchird C, Meevootisom M (1996) Cloning and nucleotide sequencing of a lipase gene from Bacillus subtilis WRRL-B558. World J Microbiol Biotechnol 12:619–623

    Article  CAS  Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Ryota Tokishige and Liu Feng for their help in determining the nucleotide sequence of the lipA gene from Bacillus sp. HH-01.

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Authors and Affiliations

  1. Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba, 263-8522, Japan

    Takashi Kamijo, Sadaharu Ema, Hiroko Hayashi & Ryo Nagata

  2. Graduate School of Advanced Integration Science, Chiba University, 648 Matsudo, Matsudo-shi, Chiba, 271-8510, Japan

    Akihiro Saito & Akikazu Ando

  3. Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba, 271-8510, Japan

    Inchi Yoh & Yoshiho Nagata

Authors
  1. Takashi Kamijo
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  2. Akihiro Saito
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  3. Sadaharu Ema
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  4. Inchi Yoh
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  5. Hiroko Hayashi
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  6. Ryo Nagata
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  7. Yoshiho Nagata
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  8. Akikazu Ando
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Correspondence to Akihiro Saito.

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Kamijo, T., Saito, A., Ema, S. et al. 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 (2011). https://doi.org/10.1007/s10482-010-9474-9

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  • DOI: https://doi.org/10.1007/s10482-010-9474-9

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