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

Background: 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. Results: ThegeneAnFaeAthatencodesatypeAferuloylesterasewassuccessfullyexpressedin Pichiapastoris bya two-copy engineered yeast. After a screen in shaker fl ask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZ α A-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZ α FA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0 – 6.5 and at below 45°C. Its activity was not signi fi cantly affected by K + , Ca 2+ , Mg 2+ , Cu 2+ , Zn 2+ , Mn 2+ , Na + and EDTA, but activated by Fe 2+ . The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions: The two-copy strain GSKZ α FA20 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 .