The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1？h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72？h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, of 0.07, and 120？h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures. 1. Introduction Carboxylic ester hydrolases (EC 3.1.1.x) (CEH) are a diverse group of hydrolases which split carboxylic acid esters in different types of molecules. Lipases (E.C. 3.1.1.3) and esterases (E.C. 3.1.1.1) are the main groups of natural biocatalysts that promote the ester bond cleavage and formation [1]. The most important feature that distinguishes lipases and esterases is the substrate specificity. Lipases preferentially hydrolyze water-insoluble esters such as triglycerides composed of long-chain fatty acids while esterases prefer short-chain acid triglycerides. Another distinction is based on protein structure, most lipases possess a hydrophobic domain (lid) covering the active site, a feature that is absent in esterases [2]. Moreover, esterases obey classical Michaelis-Menten kinetics, whereas lipases need a minimum substrate concentration before high activity is observed [3]. In this direction, esterases may offer advantages as catalysts over lipases in the absence of an interface. All aspects described above demonstrate how fundamentally significant esterases are for biotechnological processes and why the search for new biocatalysts has become very important. Enzyme immobilization ensures recycling of the biocatalyst, allows easy product separation, and may improve performance of the enzyme [4]. The
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