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The Scientific World Journal
Volume 2014, Article ID 382581, 8 pages
http://dx.doi.org/10.1155/2014/382581
Research Article

Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket of Yarrowia lipolytica Lipase Lip2 Affects Its Activity and Thermostability

Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037, Luoyu Road, Wuhan 430074, China

Received 4 June 2014; Accepted 19 July 2014; Published 13 August 2014

Academic Editor: Mehmet Yakup Arica

Copyright © 2014 Guilong Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12) to p-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.