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Construction and expression of two-copy engineered yeast of feruloyl esterase

Zhou, Min, Huang, Zhiqing, Zhou, Bo, Luo, Yanliu, Jia, Gang, Liu, Guangmang, Zhao, Hua, Chen, Xiaoling
Electronic Journal of Biotechnology 2015 v.18 pp. 338-342
Aspergillus niger, EDTA (chelating agent), Pichia pastoris, antibiotics, biotechnology, calcium, catalytic activity, copper, economic sustainability, enzyme activity, feruloyl esterase, genes, genetically engineered microorganisms, glycoproteins, industrial applications, iron, magnesium, manganese, molecular weight, pH, plasmids, potassium, secretion, sodium, zinc
Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases.The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4U/mL was obtained. Then, the pPICZαA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZαFA20 with the highest feruloyl esterase activity of 15.49U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40kDa. It displayed the maximum activity at pH6.0 and 50°C, and was stable at a pH range of 4.0–6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2+, Mg2+, Cu2+, Zn2+, Mn2+, Na+ and EDTA, but activated by Fe2+. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5mM and 69.0U/mg, respectively.The two-copy strain GSKZαFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.