Production, purification, characterization and gene cloning of an esterase produced by Aureobasidium melanogenum HN6.2

Aureobasidium melanogenum HN6.2 was found to able to produce an esterase of 208.1 +/- 2.7 units per ml within 72 h. A molecular weight of the purified esterase was 60.2 kDa and its PI was 4.86. The optimal pH and temperature of the purified esterase were 8.0 and 40 degrees C, respectively. The purified esterase was stable at the temperature less than 40 degrees C and in the pH range of 7.5-8.0. The esterase activity was greatly inhibited in the presence of Zn2+, Hg2+, Fe2+, Ni2+ and SDS. K-m and V-max values of the enzyme for rho-nitrophenyl butyrate were 68.6 mu M and 251.4 mu M per min, respectively. Mass analysis showed that it belonged to a carboxylesterase. After an esterase gene (Car-Est) cloned from the genomic DNA of the marine yeast strain HN6.2 was disrupted, the disruptant Y44 obtained exhibited a significant decrease in esterase activity. Meanwhile, a transcriptional level of the Car-Est gene of the disruptant Y44 was only 5.18% that of the Car-Est gene of its wild type strain HN6.2, confirming that the cloned esterase gene was indeed closely related to the esterase activity of the yeast HN6.2 strain. (C) 2016 Elsevier Ltd. All rights reserved.