Molecular breeding of white rot basidiomycete for efficient saccharification in biomass conversion process

白色真菌平菇（Pleuristostreatus）分泌的一种多功能过氧化物酶MnP 2具有独特的高分子量化合物氧化活性。通过含有具有定点碱基取代的重组mnp 2的糙皮侧耳转化体产生一系列突变体MnP 2。将每个突变蛋白纯化至均一，并用动力学和对低分子量和高分子量底物的反应性以及光谱方法进行表征。在这项工作中测定的所有变体都显示出与野生型MnP 2基本相似的Mn^<2+>和H_2O_2动力学性质。另一方面，证明了将暴露色氨酸残基（W170）替换为丙氨酸失去了对藜芦醇（VA）、Poly R-478和RNase A的氧化活性。此外，取代周围的氨基添加残基影响氧化的高分子量化合物，但VA。从这些结果，它清楚地表明，MnP 2氧化高分子量的化合物直接通过远程电子转移途径从W170血红素部分。本文还讨论了突变体MnP 2中碳水化合物流失的额外修饰。为建立真菌多功能过氧化物酶基因打靶系统，克隆并分析了一个与非同源末端连接系统相关的Ku 70蛋白基因。

A versatile peroxidase MnP2, secreted by a white rot fungus Pleurotus ostreatus was characterized for its unique property of high molecular weight compounds oxidizing activity. A series of mutant MnP2 were produced by P. ostreatus transformants containing a recombinant mnp2 with site-directed base substitutions. Each mutant protein was purified to homogeneity and characterized with kinetics and reactivity for low- and high-molecular weight substrates, and spectroscopic methods. All variants assayed in this work showed substantially similar kinetic properties for Mn^<2+> and H_2O_2 to those of wild type MnP2. On the other hand, it was demonstrated that substitution of an exposing tryptophan residue (W170) to alanine lost oxidation activity for veratrylacohol (VA), Poly R-478 and Rnase A. Furthermore, substitution of the surrounding amino add residues affected oxidation of the high-molecular weight compounds but VA. From these results, it was clearly demonstrated that MnP2 oxidizes high-molecular weight compounds directly via long range electron transfer pathway from W170 to heme moiety. An extra modification of carbohydrate drain in a mutant MnP2 was also discussed. This is the first report of mutational analysis of fungal versatile peroxidase produced by a homologous expression system.For the development of a gene-targeting system in basidiomycete, a gene encoding for Ku70 protein which is involved in non-homologous end joining system was cloned and analyzed.