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In Silico and Experimental Studies on the Effect of α3 and α5 Deletion on the Biochemical Properties of Bacillus thermocatenulatus Lipase

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Abstract

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni–NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively.

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Acknowledgements

Work on project number of 674 was supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB).

Funding

This work was supported by the National Research Institute for Genetic Engineering and Biotechnology (NIGEB) (Grant Number 628).

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Authors and Affiliations

  1. Department of Systems Biotechnology, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Ali Asghar Karkhane, Soha Zargoosh, Moein Aliakbari & Seyed Safa-Ali Fatemi

  2. Department of Bioprocess Engineering, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Saeed Aminzadeh

  3. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran

    Ali Asghar Karkhane

  4. Anesthesia Department of Medical College, Hamedan, Iran

    Behrouz Karkhaneh

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All authors contributed to the conduct of the study and preparation of the article. All materials were created by AAK. In silico studies by AAK, SZ, and MA. All tests were conducted by SZ, MA, SA and AAK. Paper preparation by AAK.

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Correspondence to Ali Asghar Karkhane.

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Karkhane, A.A., Zargoosh, S., Aliakbari, M. et al. In Silico and Experimental Studies on the Effect of α3 and α5 Deletion on the Biochemical Properties of Bacillus thermocatenulatus Lipase. Mol Biotechnol (2023). https://doi.org/10.1007/s12033-023-00804-0

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