Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters

International Journal of Molecular Sciences, vol. 21, pp. 1990

Abstract

The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated by rational design of enzymes. In this work, we propose a semi-rational mutational strategy focused on introducing diversity in structurally variable regions in enzymes. The identified sequences are subjected to a progressive deletion of two amino acids and the joining residues are subjected to saturation mutagenesis using NNK degenerate codons. This strategy offers a novel library diversity approach while simultaneously decreasing enzyme size in the variable regions. In this way, we intend to identify and reduce variable regions found in enzymes, probably resulting from neutral drift evolution, and simultaneously studying the functional effect of said regions. This strategy was applied to Bacillus. subtilis lipase A (BSLA), by selecting and deleting six variable enzyme regions (named regions 1 to 6) by the deletion of two amino acids and additionally randomizing the joining amino acid residues. After screening, no active variants were found in libraries 1% and 4%, 15% active variants were found in libraries 2% and 3%, and 25% for libraries 5 and 6 (n = 3000 per library, activity detected using tributyrin agar plates). Active variants were assessed for activity in microtiter plate assay (pNP-butyrate), thermal stability, substrate preference (pNP-butyrate, -palmitate), and compared to wildtype BSLA. From these analyses, variant P5F3 (F41L-ΔW42-ΔD43-K44P), from library 3 was identified, showing increased activity towards longer chain p-nitrophenyl fatty acid esters, when compared to BSLA. This study allowed to propose the targeted region 3 (positions 40–46) as a potential modulator for substrate specificity (fatty acid chain length) in BSLA, which can be further studied to increase its substrate spectrum and selectivity. Additionally, this variant showed a decreased thermal resistance but interestingly, higher isopropanol and Triton X-100 resistance. This deletion-randomization strategy could help to expand and explore sequence diversity, even in already well studied and characterized enzyme backbones such as BSLA. In addition, this strategy can contribute to investigate and identify important non-conserved regions in classic and novel enzymes, as well as generating novel biocatalysts with increased performance in specific processes, such as enzyme immobilization.

Authors 7

1. Ronny Martínez corresponding

   University of La Serena

   Affiliation as printed

   Departamento de Ingeniería en Alimentos, Instituto de Investigación Multidisciplinaria en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile

2. Claudia Bernal

   University of La Serena

   Affiliation as printed

   Departamento de Ingeniería en Alimentos, Instituto de Investigación Multidisciplinaria en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile

3. Rodrigo Álvarez

   Universidad Santo Tomás · University of La Serena

   Affiliation as printed

   Departamento de Ingeniería en Alimentos, Instituto de Investigación Multidisciplinaria en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile

   Escuela de Tecnología Médica, Facultad de Salud, Sede La Serena, Universidad Santo Tomás, La Serena 1710172, Chile

4. Christopher Concha

   University of La Serena

   Affiliation as printed

   Departamento de Ingeniería en Alimentos, Instituto de Investigación Multidisciplinaria en Ciencia y Tecnología, Universidad de La Serena, Av. Raúl Bitrán 1305, La Serena 1720010, Chile

5. Ricardo Cabrera

   University of Chile

   Affiliation as printed

   Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile

6. Gaurao V. Dhoke Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

7. Mehdi D. Davari Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany