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Expression and Biochemical Characterization of a Thermophilic Organic Solvent-Tolerant Lipase from Bacillus sp. DR90

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Abstract

The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.

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Abbreviations

HPLC:

High performance liquid chromatography

ILs:

Ionic liquids

LB:

Luria–Bertani

PMSF:

Phenylmethanesulfonyl fluoride

IPTG:

Isopropyl-b-d-thiogalactopyranoside

IMAC:

Immobilized metal ion affinity chromatography

pNPB:

p-Nitrophenyl butyrate

pNPA:

p-Nitrophenyl acetate

pNPP:

p-Nitrophenyl palmitate

pNP:

p-Nitrophenol

PPL:

Porcine pancreatic lipase

ORF:

Open reading frame

SDS-PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

EDTA:

Ethylene diaminetetraacetic acid

DTNB:

55′-Dithiobis-2-nitrobenzoic acid

DTT:

Dithiothreitol

CTAB:

Cetyltrimethylammonium bromide

[EMIm][Br]:

1-Ethyl-3-methylimidazolium bromide

[BMIm][Br]:

1-n-Butyl-3-methylimidazolium bromide

[HMIm][Br]:

1-Hexyl-3-methylimidazoliumbromide

[BMIm][Cl]:

1-Butyl-3-methylimidazolium chloride

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Acknowledgments

This work was funded by Grant number 91058286 from Iran National Science Foundation (INSF) and is gratefully acknowledged by the authors.

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Authors and Affiliations

  1. Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

    Ahmad Asoodeh, Shirin Emtenani & Shamsi Emtenani

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  1. Ahmad Asoodeh
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  2. Shirin Emtenani
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  3. Shamsi Emtenani
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Correspondence to Ahmad Asoodeh.

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Asoodeh, A., Emtenani, S. & Emtenani, S. Expression and Biochemical Characterization of a Thermophilic Organic Solvent-Tolerant Lipase from Bacillus sp. DR90. Protein J 33, 410–421 (2014). https://doi.org/10.1007/s10930-014-9574-x

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