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Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope

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Abstract

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263 T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni–NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min−1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

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Funding

This study was supported by the Gebze Technical University research project (BAP 2012 A-13).

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Gebze Technical University, Gebze, 41400, Kocaeli, Turkey

    F. İnci Özdemir, Ahmet Tülek & Davut Erdoğan

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  1. F. İnci Özdemir
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  2. Ahmet Tülek
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  3. Davut Erdoğan
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İÖ designed the study. AT, DE performed the study. AT, DE and İÖ analysed the data. İÖ and AT wrote the paper. All authors read and approved the manuscript.

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Correspondence to F. İnci Özdemir.

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Özdemir, F.İ., Tülek, A. & Erdoğan, D. Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope. Protein J 40, 436–447 (2021). https://doi.org/10.1007/s10930-021-09987-4

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