Vol.:(0123456789) 1 3 Plant Cell Tiss Organ Cult (2017) 130:13–24 DOI 10.1007/s11240-017-1200-0 ORIGINAL ARTICLE Horseradish esterases: detection, purifcation and identifcation Ivana Leščić Ašler 1  · Petra Peharec Štefanić 2  · Biljana Balen 2  · Günter Allmaier 3  · Martina Marchetti‑Deschmann 3  · Biserka Kojić‑Prodić 1   Received: 17 August 2016 / Accepted: 10 March 2017 / Published online: 19 March 2017 © Springer Science+Business Media Dordrecht 2017 Keywords Armoracia lapathifolia · Protein identifcation · Protein purifcation · Electrophoresis · Mass spectrometry · Esterase Introduction In vitro grown tissues can be used for production of biolog- ically active compounds. Esterases are known to participate in many biological processes in plants, but only a handful of plant esterases are thoroughly characterized. For many decades now, hydrolases (esterases in particular) have been studied as valuable tools for stereospecifc hydrolysis and synthesis of many biotechnologically important com- pounds. Microbial enzymes have gained interest for their wide uses in bioconversions related to food, agriculture, production of fne chemicals, medicine, diagnostics, energy and waste treatment (Singh et al. 2016; Gurung et al. 2013; Sayali and Surekha 2013). Esterases have been used in synthesis of chiral drugs, in producing favoring and fra- grance compounds, as well as for hydrolysis of milk fat for the purpose of favor enhancement in the manufacturing of cheese-related products (Panda and Gowrishankar 2005). Microbial esterases from baker’s yeast and aroma esters have been used in production of alcoholic beverages, over three decades (Suomalainen 1981). The research related to enzymatic degradation of acetyl-xylan by cooperativity of fungal esterases and xylanases, published in 1985 (Bieley et al. 1985), initiated very important studies on decom- position of biomass which is among the highest priority in biotechnology these days (Liu and Ding 2016). Bacte- rial and fungal esterases are generally well-characterized and widely applied (Borelli and Trono 2015), due to the simple access to large amounts of these enzymes (since genetic manipulation with enzymes of microbial origin is Abstract Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purifcation and identifcation of esterases from diferent horserad- ish tissues: plantlets and two tumor tissue lines. Horizon- tal IEF system enabled separation of six esterase isoforms with quite diferent pI values as well as with pronounced diferences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel elec- trophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purifcation and characteriza- tion. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in- solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identifcation of two ester- ase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1. * Ivana Leščić Ašler ilescic@irb.hr 1 Department of Physical Chemistry, Laboratory for Chemical and Biological Crystallography, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia 2 Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia 3 Institute for Chemical Technologies and Analytics, Technische Universität Wien (Vienna University of Technology), Getreidemarkt 9/164, 1060 Vienna, Austria