The enzyme aminoacylase was used to produce L-theanine from its derivative N-acetyl-DL-theanine. For stabilization purposes the aminoacylase was immobilized on polyvinyl alcohol-based nanofibrous membranes generated by electrospinning. The immobilized aminoacylase exhibited better resistance to changes in temperature and pH than the free enzyme with optimal conditions being pH 8 and 52 degrees C. Under these conditions, K-m values were two to three times higher than those of the free enzyme (3.6 mM). Thermostability was also significantly improved; the activity of the immobilized enzyme was retained at approximately 70% after 6 days at 52 degrees C. These results indicate potential applications of nanofibrous-immobilized aminoacylase for industrial production of pure L-theanine. (C) 2015 Published by Elsevier B.V.