© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 1 Introduction Recent developments of biotransformation processes have been driven by the increasing demand for sustain- able, energy-saving, and environmentally friendly syn- thetic strategies [1, 2]. Although enzymes are biocatalysts with high efficiency and excellent selectivity, their wide- spread application in industrial processes is often limited due to poor reusability and low operational stability [3]. Many immobilization strategies have been developed to improve biocatalyst properties [4–7]. Maximum retention of the enzymatic activity has been the primary concern, whereas less attention has been paid to the biocatalyst structure and its changes during immobilization and use under process conditions. This is surprising considering Research Article Microscopic monitoring provides information on structure and properties during biocatalyst immobilization Sarka Bidmanova 1,2 , Eva Hrdlickova 3 , Josef Jaros 4 , Ladislav Ilkovics 4 , Ales Hampl 2,4 , Jiri Damborsky 1,2,3 and Zbynek Prokop 1,2,3 1 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic 2 International Clinical Research Center, St. Anne’s University Hospital Brno, Czech Republic 3 Enantis Ltd., Brno, Czech Republic 4 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stabil- ity, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques – bright- field, confocal and electron microscopy – were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non- treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only fac- tor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocat- alyst during immobilization and under process conditions. Keywords: Cross-linked enzyme aggregates · Immobilization · Microscopy · Polyvinyl alcohol · Structure Correspondence: Assoc. Prof. Zbynek Prokop, Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic E-mail: zbynek@chemi.muni.cz Additional Correspondence: Prof. Jiri Damborsky, Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic E-mail: jiri@chemi.muni.cz Abbreviations: CLEAs, cross-linked enzyme aggregates; lentikats, lens- shaped particles of polyvinyl alcohol Biotechnol. J. 2014, 9 DOI 10.1002/biot.201300049 www.biotechnology-journal.com Biotechnology Journal Received 29 JAN 2014 Revised 31 JAN 2014 Accepted 14 MAR 2014 Accepted article online 17 MAR 2014 Supporting information available online