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In Silico Engineering of Enzyme Access Tunnels

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Enzyme Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2397))

Abstract

Enzyme engineering is a tailoring process that allows the modification of naturally occurring enzymes to provide them with improved catalytic efficiency, stability, or specificity. By introducing partial modifications to their sequence and to their structural features, enzyme engineering can transform natural enzymes into more efficient, specific and resistant biocatalysts and render them suitable for virtually countless industrial processes. Current enzyme engineering methods mostly target the active site of the enzyme, where the catalytic reaction takes place. Nonetheless, the tunnel that often connects the surface of an enzyme with its buried active site plays a key role in the activity of the enzyme as it acts as a gatekeeper and regulates the access of the substrate to the catalytic pocket. Hence, there is an increasing interest in targeting the sequence and the structure of substrate entrance tunnels in order to fine-tune enzymatic activity, regulate substrate specificity, or control reaction promiscuity.

In this chapter, we describe the use of a rational in silico design and screening method to engineer the access tunnel of a fructosyl peptide oxidase with the aim to facilitate access to its catalytic site and to expand its substrate range. Our goal is to engineer this class of enzymes in order to utilize them for the direct detection of glycated proteins in diabetes monitoring devices. The design strategy involves remodeling of the backbone structure of the enzyme , a feature that is not possible with conventional enzyme engineering techniques such as single-point mutagenesis and that is highly unlikely to occur using a directed evolution approach.

The proposed strategy, which results in a significant reduction in cost and time for the experimental production and characterization of candidate enzyme variants, represents a promising approach to the expedited identification of novel and improved enzymes. Rational enzyme design aims to provide in silico strategies for the fast, accurate, and inexpensive development of biocatalysts that can meet the needs of multiple industrial sectors, thus ultimately promoting the use of green chemistry and improving the efficiency of chemical processes.

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Author information

Authors and Affiliations

  1. Biomolecular Engineering Lab, Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy

    Alfonso Gautieri, Federica Rigoldi & Alberto Redaelli

  2. Center for Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, Milan, Italy

    Archimede Torretta & Emilio Parisini

  3. Latvian Institute of Organic Synthesis, Riga, Latvia

    Emilio Parisini

Authors
  1. Alfonso Gautieri
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  2. Federica Rigoldi
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  3. Archimede Torretta
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  4. Alberto Redaelli
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  5. Emilio Parisini
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Corresponding authors

Correspondence to Alfonso Gautieri or Emilio Parisini .

Editor information

Editors and Affiliations

  1. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Francesca Magnani

  2. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Chiara Marabelli

  3. Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland

    Francesca Paradisi

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Gautieri, A., Rigoldi, F., Torretta, A., Redaelli, A., Parisini, E. (2022). In Silico Engineering of Enzyme Access Tunnels. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1826-4_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1826-4_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1825-7

  • Online ISBN: 978-1-0716-1826-4

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