Immobilized Biocatalyst Engineering: High throughput enzyme immobilization for the integration of biocatalyst improvement strategies Karen Rodríguez-Núñez a , Claudia Bernal a,b, ⁎, Ronny Martínez a, ⁎⁎ a Laboratorio de Tecnología de Enzimas para Bioprocesos, Departamento de Ingeniería en Alimentos, Universidad de La Serena, Av. Raúl Bitrán 1305, 1720010 La Serena, Chile b Instituto de Investigación Multidisciplinario en Ciencia y Tecnología, Universidad de La Serena, Benavente 980, 1720010 La Serena, Chile abstract article info Article history: Received 22 October 2020 Received in revised form 5 December 2020 Accepted 12 December 2020 Available online xxxx Keywords: High-throughput immobilization Immobilized Biocatalyst Engineering Integrated screening Protein engineering Enzyme immobilization Bacillus subtilis lipase A The increasing use of sustainable manufacturing technologies in the industry presents a constant challenge for the development of suitable biocatalysts. Traditionally, improved biocatalysts are developed either using protein engineering (PE) or enzyme immobilization (EI). However, these approaches are usually not simultaneously ap- plied. In this work, we designed and validated an enzyme improvement platform, Immobilized Biocatalyst Engi- neering (IBE), which simultaneously integrates PE and EI, with a unique combination of improvement through amino acid substitutions and attachment to a support material, allowing to select variants that would not be found through single or subsequent PE and EI improvement strategies. Our results show that there is a significant difference on the best performing variants identified through IBE, when compared to those that could be identi- fied as soluble enzymes and then immobilized, especially when evaluating variants with low enzyme as soluble enzymes and high activity when immobilized. IBE allows evaluating thousands of variants in a short time through an integrated screening, and selection can be made with more information, resulting in the detection of highly stable and active heterogeneous biocatalysts. This novel approach can translate into a higher probability of find- ing suitable biocatalysts for highly demanding processes. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Biocatalysis has emerged as an important technology development area to meet the growing demand of industrial processes [1]; requiring biocatalysts with high activity, cost effective production and purifica- tion, combined with high specificity and operational stability [2,3]. Tra- ditionally, biocatalytic processes were usually designed around enzyme capabilities and limitations; today, the trend is aimed at adapting them to the requirements of the process where they will be applied [4]. Indus- trial applications such as the resolution of racemic mixtures, synthesis, degradation or modification of molecules, demand new features that must be obtained through different improvement strategies such as protein engineering and enzyme immobilization [5,6]. Protein engi- neering seeks to improve enzymes by modifying their intrinsic proper- ties through amino acid substitutions, by which a specific functional change is achieved, using molecular tools in protein design, to generate genetic diversity, and to detect improvements in the new enzyme, in terms of activity, thermostability, resistance to organic solvents, substrate specificity and regioselectivity [2,5]. Identifying the best vari- ants present in the generated mutant library through a well-established screening system is crucial challenges of protein engineering campaigns [6,7]. Through enzyme immobilization, a soluble enzyme is confined in a space to generate an insoluble and reusable biocatalyst, maintaining the catalytic activity [8], and increasing its thermal stability and resis- tance to organic solvents [8,9]. And one of its main challenges is its lack of general conditions applicable to any enzyme a priori, which makes it difficult to extrapolate it as an industrial process [6,10,11]. Both protein engineering and enzyme immobilization are quite comple- mentary disciplines and neither of them manages appears to solve all the challenges of enzyme improvement on its own [5,6]. Protein engineering and enzyme immobilization processes are mainly used individually, and recently sequentially, whereby a strategy of mutagenesis, screening and selection of the best variant is applied, and then, this single variant with increased performance against the chosen challenge, is immobilized [12,13]. However, these enzymes are not really optimized as immobilized biocatalysts, and therefore their performance as such is not guaranteed [6]. Both strategies aim to ac- complish the same goal: to improve biocatalyst performance. But both have challenges to solve, which require rethinking how to apply them. Faced with this scenario, this work presents for the first time -according to our best knowledge- the design and implementation of International Journal of Biological Macromolecules 170 (2021) 61–70 ⁎ Correspondence to: C. Bernal, Instituto de Investigación Multidisciplinario en Ciencia y Tecnología, Universidad de La Serena, Benavente 980, 1720010 La Serena, Chile. ⁎⁎ Corresponding author. E-mail addresses: cbernal@userena.cl (C. Bernal), remartinez@userena.cl (R. Martínez). https://doi.org/10.1016/j.ijbiomac.2020.12.097 0141-8130/© 2020 Elsevier B.V. All rights reserved. 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