靈芝屬菌株錳過氧化酶的選殖與表達

Abstract

靈芝屬(Ganoderma spp.)是一種白腐型真菌，具有各種可分解木材的外泌酵素，包括纖維素水解酵素(cellulase)和半纖維素水解酵素(hemicellulase),、果膠質水解酵素(pectinase)、漆氧化酶(laccase)、木質素過氧化酶(lignin peroxidase)和錳過氧化酶(manganese peroxidase)。其中錳過氧化酶是一種含有血基質(heme)的酵素，在二價錳離子以及過氧化氫存在的環境中能將酚類多環物質氧化分解，白腐型真菌利用此酵素分解木質素結構使其菌絲能有效深入木質纖維中分解纖維素等多醣以獲得養分。本實驗以含有ABTS的培養皿測試十株靈芝屬菌株的胞外酵素，發現所測試靈芝屬真菌都有可分解ABTS(2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid))的外泌酵素。並利用Genome Walking、五端RACE和PCR增幅技術自台灣靈芝(G. formosanum 0109, BZ)、新日本靈芝(G. neo-japonicum 0813, VZ)和南方靈芝(G. australe 0705)中選殖出六段新的錳過氧化酶基因序列。以RT-PCR增幅基因首尾端具有限制酶切位的台灣靈芝0109錳過氧化酶的cDNA，並將此基因轉殖入大腸桿菌(Escherichia coli)以及嗜甲醇畢赤氏酵母(Pichia methanolica)進行異源表達，得到在C端接有六個組胺酸(histidine)的錳過氧化酶以利後續純化的重組蛋白。

Lignin, component of plant secondary cell wall, binds to hemicellulose by covalent bonds and defends bacteria or fungi invading into the core of the wood. Lignin is a complicated polymer composed of aromatic compounds, which are ferulic acid, sinapic acid and p-coumaric acid etc. In paper pulping industry, lignin is hard-degradable and influences the brightness of paper. Generally, people use concentrated acid or alkali to precipitate or dissolve lignin. In recent study, the paper would be brighter and whiter by using manganese peroxidase in pulping procedure. Manganese peroxidase, which was first isolated from white rot fungi Phanerochaete chrysosporium by Glenn and Gold in 1985, belongs to heme-containing peroxidase family. This extracellular enzyme is a glycoprotein with five disulfide bonds in general and contains two calcium ions in the structure. When it catalyses the substrates, hydrogen peroxide which produced by glyoxal oxidase or aryl-alcohol oxidase is necessary as electron donor. Mn(II) would be oxidized to Mn(III) by heme-oxygen radical complex. Mn(III) cations would be stabilized by alfa-hydroxyl acids, such as succinic acid or lactic acid, and oxidize lignin or lignin-like compounds. Manganese peroxidase has been found in many of white rot fungi, including Trametes versicolor, Pleurotus ostreatus, Armillaria mellea, etc, however, the studies of manganese peroxidase from Ganoderma spp. are few and most of them were focus on preotein level. The only published cDNA sequence was from G. applanatum. In this study, we cloned six new sequences of manganese peroxidase from G. formosanum, G. neo-japonicum and G. australe. Furthermore, we expressed the manganese peroxidase of G. formosanum in Escherichia coli and Pichia methanolica and produced recombinant enzyme.