Skip to main content
SpringerLink
Log in

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.

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

Similar content being viewed by others

References

  • Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(Pt 1):177–183

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Balan A, Ibrahim D, Abdul Rahim R, Ahmad Rashid FA (2012) Purification and characterization of a thermostable lipase from Geobacillus thermodenitrificans IBRL-nra. Enzyme Res

  • Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and applications in biocatalysis. FEMS Microbiol Rev 26:73–81

    Article  CAS  PubMed  Google Scholar 

  • Carrasco-Lopez C, Godoy C, de las Rivas B, Fernandez-Lorente G, Palomo JM, Guisan JM, Fernandez-Lafuente R, Martinez-Ripoll M, Hermoso JA (2009) Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements. JBC 284(7):4365–4372

    Article  CAS  Google Scholar 

  • Charbonneau DM, Meddeb-Mouelhi F, Beauregard M (2010) A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: evidence for a new carboxylesterase family. J Biochem 148(3):299–308

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Dosztanyi Z, Fiser A, Simon I (1997) Stabilization centers in protein: identification, characterization and predictions. J Mol Biol 272:597–612

    Article  CAS  PubMed  Google Scholar 

  • Dosztanyi Z, Magyar C, Tusnady GE, Simon I (2003) SCide: identification of stabilization centers in proteins. Bioinformatics 19:899–900

    Article  CAS  PubMed  Google Scholar 

  • Ebrahimpour A, Rahman RNZRA, Basri M, Salleh AB (2011) High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM. Bioresour Technol 102:6972–6981

    Article  CAS  PubMed  Google Scholar 

  • Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive sequence similarity searching. Nucleic Acids Res 39:29–37

    Article  Google Scholar 

  • Ghori MI, Iqbal MJ, Hameed A (2011) Characterization of a novel lipase from Bacillus sp. isolated from tannery wastes. Braz J Microbiol 42:22–29

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gomes N, Braga A, Teixeira JA, Belo I (2013) Impact of lipase-mediated hydrolysis of castor oil on γ-decalactone production by Yarrowia lipolytica. J Am Oil Chem Soc 90:1131–1137

    Article  Google Scholar 

  • Guncheva M, Zhiryakova D (2011) Catalytic properties and potential applications of Bacillus lipases. J Mol Catal B Enzym 68:1–21

    Article  CAS  Google Scholar 

  • Hasan F, Shah AA, Hameed A (2006) Industrial application of microbial lipases. Enzyme Microb Technol 39:235–251

    Article  CAS  Google Scholar 

  • Hasan F, Shah AA, Javed S, Hameed A (2010) Enzymes used in detergents: lipases. Afr J Biotechnol 9(31):4836–4844

    CAS  Google Scholar 

  • Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:545–549

    Article  Google Scholar 

  • Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397

    Article  CAS  PubMed  Google Scholar 

  • Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tolls for biotechnology. Tibtech 16:396–403

    Article  CAS  Google Scholar 

  • Jeong ST, Kim HK, Kim SJ, Chi SW, Pan JG, Oh TK, Ryu SE (2002) Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase. J Biol Chem 277(19):17041–17047

    Article  CAS  PubMed  Google Scholar 

  • Jiang Y, Zhou X, Chen Z (2010) Cloning, expression, and biochemical characterization of a thermostable lipase from Geobacillus stearothermophilus JC. World J Microbiol Biotechnol 26:747–751

    Article  CAS  Google Scholar 

  • Katata L, Nagaraju V, Crouch AM (2006) Determination of ethylenediaminetetraacetic acid, ethylenediaminedisuccinic acid and iminodisuccinic acid in cosmetic products by capillary electrophoresis and high performance liquid chromatography. Anal Chim Acta 579:177–184

    Article  CAS  PubMed  Google Scholar 

  • Kim HK, Park SY, Lee JK, Oh TK (1998) Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci Biotechnol Biochem 62:66–71

    Article  CAS  PubMed  Google Scholar 

  • Kuisiene N, Raugalas J, Stuknyte M, Chitavicius D (2007) Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer. FEMS Microbiol Lett 277:165–172

    Article  CAS  PubMed  Google Scholar 

  • Kwon DY, Rhee JS (1986) A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J Am Oil Chem Soc 63:89–92

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:380–685

    Google Scholar 

  • Lee DW, Koh YS, Kim KJ, Kim BC, Choi HJ, Kim DS, Suhartono MT, Pyun YR (1999) Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol Lett 179:393–400

    Article  CAS  PubMed  Google Scholar 

  • Leow TC, Rahman RNZRA, Basri M, Salleh AB (2004) High level expression of thermostable lipase from Geobacillus sp. strain T1. Biosci Biotechnol Biochem 68(1):96–103

    Article  CAS  PubMed  Google Scholar 

  • Leow TC, Rahman RNZRA, Basri M, Salleh AB (2007) A thermoalkaliphilic lipase of Geobacillus sp. T1. Extremophiles 11:527–535

    Article  CAS  PubMed  Google Scholar 

  • Levisson M, van der Oost J, Kengen SWM (2007) Characterization and structural modelling of a new type of thermostable esterase from Thermotoga maritime. FEBS J 274:2832–2842

    Article  CAS  PubMed  Google Scholar 

  • Li H, Zhang X (2005) Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1. Protein Expr Purif 42:153–159

    Article  PubMed  Google Scholar 

  • Magyar C, Gromiha MM, Pujadas G, Tusnady GE, Simon I (2005) SRide: a server for identifying stabilizing residues in proteins. Nucleic Acids Res 33:303–305

    Article  Google Scholar 

  • Matsumura H, Yamamoto T, Leow TC, Mori T, Salleh AB, Basri M, Inoue T, Kai Y, Rahman RNZRA (2007) Novel cation-π interaction revealed by cristal structure of thermoalkalophilic lipase. Proteins 70:592–598

    Article  Google Scholar 

  • Nardini M, Dijkstra BW (1999) Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 9(6):732–737

    Article  CAS  PubMed  Google Scholar 

  • Olechnovič K, Kulberkytė E, Venclovas Č (2012) CAD-score: a new contact area difference-based function for evaluation of protein structural models. Proteins

  • Pei J, Kim BH, Grishin NV (2008) PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res 36(7):2295–2300

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Petersen TN, Brunak S, Heijne G, Nielsen H (2011) SignalIP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786

    Article  CAS  PubMed  Google Scholar 

  • Salleh AB, Rahim ASMA, Rahman RNZRA, Leow TC, Basri M (2012) The role of Arg157Ser in improving the compactness and stability of ARM lipase. J Comput Sci Syst Biol 5(2):039–046

    CAS  Google Scholar 

  • Sambrook J, Rusell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Lab Press, New York

    Google Scholar 

  • Sangeetha R, Arulpandi I, Geetha A (2011) Bacterial lipases as potential industrial biocatalysts: an overview. Res J Microbiol 6(1):1–24

    Article  CAS  Google Scholar 

  • Shariff FM, Rahman RNZRA, Basri M, Salleh AB (2011) A newly isolated thermostable lipase from Bacillus sp. Int J Mol Sci 12:2917–2934

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sharma D, Sharma B, Shukla AK (2011) Biotechnological approach of microbial lipase: a review. Biotechnology 10(1):23–40

    Article  CAS  Google Scholar 

  • Sinchaikul S, Sookkheo B, Phutrakul S, Pan FM, Chen ST (2001) Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization. Protein Expr Purif 22:388–398

    Article  CAS  PubMed  Google Scholar 

  • Singh R, Gupta N, Goswami VK, Gupta R (2006) A simple activity staining protocol for lipases and esterases. Appl Microbiol Biotechnol 70:679–682

    Article  CAS  PubMed  Google Scholar 

  • Tayyab M, Rashid N, Akhtar M (2011) Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S: cloning and characterization of the lipase. J Biosci Bioeng 111(3):272–278

    Article  CAS  PubMed  Google Scholar 

  • Tyndall JDA, Sinchaikul S, Fothergill-Gilmore LA, Taylor P, Walkinshaw MD (2002) Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1. J Mol Biol 323:859–869

    Article  CAS  PubMed  Google Scholar 

  • Vehlow C, Stehr H, Winkelmann M, Duarte JM, Petzold L, Dinse J, Lappe M (2011) CMView: interactive contact map visualization and analysis. Bioinformatics 27(11):1573–1574

    Article  CAS  PubMed  Google Scholar 

  • Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65(1):1–43

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Villeneuve P, Muderhwa JM, Graille J, Haas MJ (2000) Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J Mol Catal B Enzym 9:113–148

    Article  CAS  Google Scholar 

  • Wahab RA, Basri M, Rahman MBA, Rahman RNZRA, Salleh AB, Leow TC (2012a) Combination of oxyanion Gln114 mutation and medium engineering to influence the enantioselectivity of thermophilic lipase from Geobacillus zalihae. Int J Mol Sci 13:11666–11680

    Article  PubMed Central  PubMed  Google Scholar 

  • Wahab RA, Basri M, Rahman MBA, Rahman RNZRA, Salleh AB, Leow TC (2012b) Engineering catalytic efficience of thermophilic lipase from Geobacillus zalihae by hydrophobic residue mutation near the catalytic pocket. ABB 3:158–167

    Article  Google Scholar 

  • Wiederstein M, Sippl MJ (2007) ProSA-web:interactive web service for the recognition of errors in three-dimensional structures of protein. Nucleic Acids Res 35:407–410

    Article  Google Scholar 

  • Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670

    CAS  PubMed Central  PubMed  Google Scholar 

  • Wu L, Liu B, Hong Y, Sheng D, Shen Y, Ni J (2010) Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase. Biotechnol Lett 32:107–112

    Article  PubMed  Google Scholar 

  • Yang Z, Zhang Y, Shen T, Xie Y, Mao Y, Ji C (2013) Cloning, expression and biochemical characterization of a novel, moderately thermostable GDSL family esterase from Geobacillus thermodenitrificans T2. J Biosci Bioeng 115(2):133–137

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformat 9:40

    Article  Google Scholar 

  • Zuo K, Zhang L, Yao H, Wang J (2010) Isolation and functional expression of a novel lipase gene isolated directly from oil-contaminated soil. Biochimica Polonica Acta 57(3):305–311

    CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the MITA (Agency of Science, Innovation and Technology) program “Development of industrial biotechnology in Lithuania 2011-2013”, project “Innovative tools for cosmetic industry (COSMETIZYM)”, Grant No. MITA 31V-18.

Author information

Authors and Affiliations

  1. Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania

    Renata Gudiukaitė, Audrius Gegeckas & Donaldas Citavicius

  2. Institute of Biotechnology, Vilnius University, Graičiūno 8, 02241, Vilnius, Lithuania

    Darius Kazlauskas

Authors
  1. Renata Gudiukaitė
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Audrius Gegeckas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Darius Kazlauskas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Donaldas Citavicius
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Renata Gudiukaitė.

Additional information

Communicated by H. Atomi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 48 kb)

Supplementary material 2 (DOC 34 kb)

Supplementary material 3 (DOC 44 kb)

792_2013_605_MOESM4_ESM.tif

Supplementary material 4 (TIFF 1566 kb) 3D structure of GD-95 lipase. The structure predicted with I-TASSER server and image wasgenerated using Pymol (DeLano Scientific, Palo Alto, CA, USA). The arrows indicate beginning of N-and C -ends, α13 helix and catalytic amino acids of GD-95 lipase; 20 C-terminal amino acids are marked in black234x176mm (300 x 300 DPI)

792_2013_605_MOESM5_ESM.tif

Supplementary material 5 (TIFF 3697 kb) Multiple alignment of 20 C-terminal amino acids from various bacterial lipases176x234mm (300 x 300 DPI)

792_2013_605_MOESM6_ESM.tif

Supplementary material 6 (TIFF 16860 kb) Effect of pH on the stability of GD-95 lipase and its modified variants. a: finely dotted line – Pre-GD-95; straight line - GD-95. b: finely dotted line – Pre-GD-95; straight line – Pre-GD-95-10; coarsely dottedline - GD- 95-10. The remaining activity was assayed under standard assay conditions after the purified recombinant lipases were pre-incubated at various pHs (1:4) at room temperature for 30 min. A specific activity of 270.0U/mg (GD-95), 100.7U/mg (Pre-GD-95), 113.0U/mg (GD-95-10), 32.3U/mg (Pre-GD-95-10) was recorded as 100%176x234mm (300 x 300 DPI)

792_2013_605_MOESM7_ESM.tif

Supplementary material 7 (TIFF 1933 kb) SDS–PAGE (12%) analysis of recombinant Pre-GD-95 (a), GD-95-10 (c) and GD-95 (e) lipasespurified using affinity chromatography. M - PageRulerTM Unstained Protein Ladder. The arrows indicate thetarget proteins. Purification of Pre-GD-95 lipase (a): lane 1 - extracellular crude samples of enzyme; lane 2 -flow through; lane 3 - washing fraction; lanes 4–10 - 1-7 elution fraction, respectively. Purification of GD-95-10 lipase (c): lanes 1-5 - elution fraction; purification of GD-95 lipase (e): lanes 1-5 - elution fraction. band d - zymogram of purified Pre-GD-95 (b), GD-95 (in d Lane 1), Pre-GD-95-10 (in d Lane 3) and Pre-GD-95-20 (in d Lane 2) lipases. Zymogram results with GD-95-10 and GD-95-20 not shown234x176mm (300 x 300 DPI)

792_2013_605_MOESM8_ESM.tif

Supplementary material 8 (TIFF 1285 kb) Effect of pH on the enzyme activity of GD-95 lipase and its modified variants. a: finely dotted line –Pre-GD-95; straight line - GD-95. b: finely dotted line – Pre-GD-95; straight line – Pre-GD-95-10; coarselydotted line - GD-95-10. c: finely dotted line –Pre-GD-95; straight line – Pre-D-95-20; coarsely dotted line -GD-95-20. Enzyme activity was assayed under standard enzyme assay conditions. A specific activity of220.3U/mg (GD-95), 102.4U/mg (Pre-GD-95), 195.6U/mg (GD-95-10), 32.3U/mg (Pre-GD-95-10),2.7U/mg (Pre-GD-95-20), 5.1U/mg (GD-95-20) was recorded as 100% at pH 9176x234mm (300 x 300 DPI)

792_2013_605_MOESM9_ESM.tif

Supplementary material 9 (TIFF 16857 kb) Arrhenius plot for the determination of activation energy of Pre-GD-95 and GD-95 lipases. Thesquare marks Pre-GD-95, rhombs - GD-95 lipases. T is Kelvin temperature234x176mm (300 x 300 DPI)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gudiukaitė, R., Gegeckas, A., Kazlauskas, D. et al. Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95. Extremophiles 18, 131–145 (2014). https://doi.org/10.1007/s00792-013-0605-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-013-0605-x

Keywords

Search

Navigation