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

[摘要] English: The xylanase from Thermomyces lanuginosus (XynA) was cloned into twoshuttle vectors, pRS416 (single copy vector) and pRS426 (multi-copy vector)adjacent to a PDC1 promoter (designated pRS416:XynA and pRS426:XynA). Anexpression cassette for this xylanase was constructed by cloning of the XynAgene into a modified a-agglutinin (Aga1) gene from Saccharomyces cerevisiae.This modification entailed the deletion of the binding domain coding region of theAga1, 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 wascloned into two shuttle vectors (pRS416 and pRS426), flanking the PDC1promoter (designated pRS416:Aga1::XynA and pRS426:Aga1::XynA). The aimof the cassette was to immobilize the expressed enzyme on the cell surface ofthe 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 lowactivity [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 yieldedactivities 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 secretedrecombinant xylanase (pRS416:Aga1::XynA and pRS426:Aga1::XynA) yielded an optimum temperature of 70 °C and an optimum pH of 6.0-7.0. Thermalstability for the recombinant xylanase was determined for temperatures 50 °C, 60°C and 70 °C, and the activation energy for pRS416:Aga1::XynA andpRS426:Aga1::XynA were calculated as 34.86 kJ/mol and 53.59 kJ/molrespectively.

[发布机构] University of the Free State