Phanerochaete chrysosporium ATCC 24725 之木質素過氧化氫酶H8基因的選殖與異源表現

Abstract

The exploitation of fossil fuels have rapidly grown since the middle of twentieth century. To today, excessive use of fossil fuels by human beings not only caused greenhouse effect and serious environmental pollution but also gradually exhaust its deposits in the world. So, it has become a top priority to search for clean and renewable alternative energy sources, the development bioenergy is one of them. Bioenergy is renewable energy made available from materials derived from biological source, lignocellulose is particularly well-suited for production because of it is the most abundant carbohydrate on the earth and rich in agricultural and forestry plants and their waste. Lignocellulose is composed of cellulose, hemicellulose and lignin, the aggregates of cellulose and hemicellulose are surrounded by rigid and hard- decomposed lignin that hinder cellulose and hemicellulose to be utilized. No better than the white rot fungi especially Phanerochaete chrysosporium can produce ligninase to decompose lignin completely and efficiently in the microbial world. Clostridium xylanolyticum Ter3 is an isolate of our lab that not only can synthesize cellulase and hemicellulase to decompose cellulose and hemicellulose, but also produce H2 and ethanol very well. In order to let C. xylanolyticum Ter3 utilize lignocellulose to produce H2 and ethanol efficiently, an aim of this study is to clone and express the ligninase gene (lip H8 gene) of P. chrysosporium in C. xylanolyticum Ter3. The lip H8 gene was also tried to express in E. coli and Pichia pastoris, owing to as a host they have many advantages such as ease of culture, rapid growth, and high-level expression of a cloned gene. By using the shuttle vector pIMPI of E. coli and Clostridium spp. to clone the lip H8 gene, recombinant plasmid pIMPI-lac+lip was constructed, but the following effort to transform this recombinant plasmid to C. xylanolyticum Ter3 by electroporation was unsuccessful, it seems that C. xylanolyticum Ter3 contained unknown restriction enzyme system to digest pIMPI-lac+lip. Two methods was used to measure the LiP H8 activity in this study:VA and ABTS assay. The highest LiP H8 activity expressed by P. chrysosporium was 50 U/L by VA assay and 101.83 U/L by ABTS assay. When the lip H8 gene was cloned into expression vector pET43.1a(+) to form recombinant plasmid pET43.1a-lip and transformed to E. coli BL21 (DE3), a 108-kDa fusion protein with Nus-tag linking to the N terminal of LiP H8 enzyme protein was produced, the highest enzyme activity shown by this fusion protein was 0.56 U/L by VA assay. The lip H8 gene was cloned into vector pPICZαA to form recombinant plasmid pPICZαA -mlip, when it was transformed to P. pastoris, the highest LiP H8 activity of 1.04 U/L was measured by VA assay, but no fusion protein with correct size was found by SDS-PAGE.