Rapid Colorimetric Assay and Yeast Surface Display for Screening of Highly Functional Fungal Lignin Peroxidase

Abstract

Rapid screening method using color reaction was developed to quantitatively evaluate the lignin peroxidase (LiP) activity. For 2, 4-dichlorophenol (DCP) degradation activity, this method correlated well with the HPLC method. The lignin peroxidase H2 gene was isolated from Phanerochaete chrysosporium, a white-rot fungus. The molecular weight of the recombinant LiP was ca. 38 kDa, which corresponded to the predicted mature LiP H2 gene. We performed cloning into pYD vector for yeast cell surface display of the gene. The surface expression was checked by the rapid, colorimetric screening method. The supernatant did not change the color, but the washed yeast cells changed the color from white to red, which confirmed the enzymes were located on the cell surface. Five colonies expressing the LiP H2 gene at high levels were selected. The average amount of 2, 4-DCP degradation was about 23%. It was approximately 63% degradation level of the LiP from the wild type fungus. This screening and selection protocol is being applied to the DNA shuffling effort to screen LiPs with improved functionality and stability.