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Identification and Characterization of a Novel Thermophilic, Organic Solvent Stable Lipase of Bacillus from a Hot Spring

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Lipids

Abstract

A novel lipase gene lip256 was cloned and identified from the genomic library of hot spring strain Bacillus sp. HT19. The deduced amino acid sequence of lip256 has less than 32% identity to a predicted esterase (Cog1752) from Photobacterium leiognathi lrivu.4.1 and contains a novel motif (GTSAG) that differs from other clusters in the lipase superfamily. Following purification, a single band was obtained with a molecular mass of 33 kDa by SDS-PAGE, and the optimal temperature and pH for lipolytic activity of Lip25 were 70 °C and 9.0, respectively. Lip256 exhibited high activity at high temperatures, with 40% maximum activity at 80 °C and good stability at temperatures ranges between 50 and 80 °C. Additionally, the enzyme was highly stable in the presence of butyl-alcohol, glycerol, acetonitrile, pyridine, and urea. However, the presence of acetone, methanol, trichloromethane, petroleum ether, hexane, tert-butanol, isopropanol, dithiothreitol, ethylenediaminetetraacetic acid, polyhexamethylene biguanide, dimethyl sulfoxide, benzene, Triton X-100, Tween-20, Tween-80, and sodium dodecyl sulfate suppressed or absolutely inhibited enzyme activity. Furthermore, Ca2+, Mg2+, and Cu2+ suppressed enzyme activity, whereas Na+, Fe3+, K+, Fe2+, and Sr2+ enhanced enzyme activity. The unique characteristics of novel lipase Lip256, including its thermo-alkaliphilic performance, high tolerance toward metal ions, inhibitors, and detergents, and high stability in organic solvents, implied that this enzyme might be an interesting candidate for industrial processes.

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Abbreviations

DMSO:

Dimethylsulfoxide

DTT:

Dithiothreitol

EDTA:

Ethylene diaminetetraacetic acid

LB:

Luria–Bertani

IPTG:

Isopropyl-b-d-thiogalactopyranoside

pHMB:

Polyhexamethylene biguanidine hydrochloride

p-NPP:

p-Nitrophenylpalmitate

ORF:

Open reading frame

SDS:

Sodium dodecyl sulphate

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Acknowledgements

This study was financially supported by Basic Scientific Research Funds of FIO, SOA (2014T09) and key lab of marine bioactive substance and modern analytical technique, SOA (MBSMAT-2015-06).

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Authors and Affiliations

  1. Key Lab of Marine Bioactive Substances, The First Institute of Oceanography, SOA, Qingdao, 266061, China

    Jiang Li & Xiumeng Liu

  2. Key Lab of Marine Bioactive Substances of Qingdao, Qingdao, 266061, China

    Jiang Li & Xiumeng Liu

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  1. Jiang Li
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  2. Xiumeng Liu
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Correspondence to Jiang Li.

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Li, J., Liu, X. Identification and Characterization of a Novel Thermophilic, Organic Solvent Stable Lipase of Bacillus from a Hot Spring. Lipids 52, 619–627 (2017). https://doi.org/10.1007/s11745-017-4265-y

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  • DOI: https://doi.org/10.1007/s11745-017-4265-y

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