Abstract
A cell surface displayed system in Pichia pastoris GS115 was developed by using GCW61, a glycosylphosphatidylinositol-modified cell wall protein from P. pastoris, as the anchor protein. Thermomyces lanuginosus lipase (TLL) was successfully displayed on the P. pastoris cell wall by fusing GCW61 gene with TLL2 gene (NCBI Accession: O59952) that was optimized with codon bias and synthesized. Cell surface displayed TLL2 was confirmed by the immunofluorescence microscopy. Flask fermentation was performed for 144 h with lipase activity up to 1964.76 U/g. Enzymatic properties of cell surface displayed TLL2 were also investigated. Displayed TLL2 occurred the maximum activity at pH 9 and 55°C and demonstrated characteristics of wide thermal adaptability and alkaline pH resistance. The optimum substrate was p-nitrophenyl hexanoate. Bivalent metal ions Ca(2+), Mn(2+), and Zn(2+) had the activation effect on displayed TLL2, while Cu(2+), Fe(2+), Fe(3+), K(+), Li(+), Na(+), and Co(2+) ions had the inhibitory effect on it. Since cell surface displayed TLL2 required less purification steps compared with free enzyme and showed high enzyme activities, it would be able to be further applied in various potential applications.