Robust enzyme design: Bioinformatic tools for improved protein stability

Abstract

The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

Immobilizing Systems Biocatalysis for the Selective Oxidation of Glycerol Coupled to In Situ Cofactor Recycling and Hydrogen Peroxide Elimination

“…With this aim, protein engineering has been shown as one of the most effective approaches to adapt enzymes to industrial requirements. [4,5] Nevertheless, the protein engineering approach cannot address the solubility issue of enzymes that hampers their reusability and workability in flow processes. Hence, protein immobilization appears as a complementary approach to protein engineering to make enzymes suitable for the chemical industry.…”

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

“…Recently, many protein tertiary structures have been determined by X-ray crystallography and NMR spectroscopy, leading to bioinformatics tools more powerful and reliable for analyzing the structures of biomacromolecules. 13 In a previous study, we successfully employed the SWISS-MODEL program to model the three-dimensional structure, the NAMD and VMD to perform molecular dynamics (MD), and the PredictProtein server programs to calculate the secondary structure, solvent accessibility and the interior hydrophobicity of aspartyl aminopeptidase from A. oryzae 3.042. 8 Here, we aim to elucidate the biochemical properties and salt-tolerant mechanisms of AP using a non-salt-tolerant neutral protease I (NP I) as control, which was also previously isolated from A. oryzae 3.042.…”

Study of Functional and Allosteric Sites in Protein Superfamilies

“…It has been shown that a bioinformatic analysis of the evolutionary relationships in functionally diverse protein superfamilies can be used not just to detect the key “hotspots” in enzyme structures, but also determine the specific amino acid substitutions to produce mutants with improved properties [54, 77-79]. Use of computational approaches in protein design has been recently reviewed [80-82]. The whole-genome sequencing of bacterial pathogens, including Mycobacterium tuberculosis [83], marked the beginning of computer genomics in medicine.…”