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

Abstract

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: 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 2.4 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 50ºC, and was stable at a pH range of 4.0-6.5 and at below 45ºC. Its activity was not significantly affected by K⁺, Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Na⁺ and EDTA, but activated by Fe²⁺. The K_m and V_max towards 4-Nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively.

Conclusions: 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 Pichia pastoris.