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
two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl
esterase activity of 2.4 U/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 15.49 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 significantly affected by K+
and EDTA, but activated by Fe2+
The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/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