Cloning of the XynA gene from Thermomyces lanuginosus and expression in Saccharomyces cerevisiae

The xylanase from Thermomyces lanuginosus (XynA) was cloned into two shuttle vectors, pRS416 (single copy vector) and pRS426 (multi-copy vector) adjacent to a PDC1 promoter (designated pRS416:XynA and pRS426:XynA). An expression cassette for this xylanase was constructed by cloning of the XynA gene into a modified a-agglutinin (Aga1) gene from Saccharomyces cerevisiae. This modification entailed the deletion of the binding domain coding region of the Aga1, and the cloning of the XynA gene into this deleted binding domain region, which is flanked by a stalk-like protein coding region. This fusion protein was cloned into two shuttle vectors (pRS416 and pRS426), flanking the PDC1 promoter (designated pRS416:Aga1::XynA and pRS426:Aga1::XynA). The aim of the cassette was to immobilize the expressed enzyme on the cell surface of the yeast cell with the expression of the xylanase on the stalk of the Aga1, however, extracellular secretion of the enzyme was obtained upon expression. Enzyme assays performed on pRS416:XynA and pRS426:XynA yielded very low activity [0.1505 U/ml (2.5088 nKat/ml) and 0.0909 U/ml (1.5153 nKat/ml) respectively], whereas pRS416:Aga1::XynA and pRS426:Aga1::XynA yielded activities of 1.7035 U/ml (28.3973 nKat/ml) and 1.7319 U/ml (28.8707 nKat/ml) respectively. The partial characterization of this extracellular secreted recombinant xylanase (pRS416:Aga1::XynA and pRS426:Aga1::XynA) yielded an optimum temperature of 70 °C and an optimum pH of 6.0-7.0. Thermal stability for the recombinant xylanase was determined for temperatures 50 °C, 60 °C and 70 °C, and the activation energy for pRS416:Aga1::XynA and pRS426:Aga1::XynA were calculated as 34.86 kJ/mol and 53.59 kJ/mol respectively.