De Novo Computational Design of a Lipase with Hydrolysis Activity towards Middle-Chained Fatty Acid Esters
Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial appli...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-05-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/10/8581 |
| Summary: | Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the <i>theozyme</i> constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta “inside-out” protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD<sub>1</sub> exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of <i>theozyme</i> intact. The redesigned lipase 1a8uD<sub>1</sub>–M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate—3.34 times higher than that of 1a8uD<sub>1</sub>. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD<sub>1</sub> and the redesigned 1a8uD<sub>1</sub>–M8 were devised from scratch. More importantly, the designed 1a8uD<sub>1</sub>–M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions. |
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| ISSN: | 1661-6596 1422-0067 |