LIGNIN BIODEGRADATON--LIGNINASE & GLUCOSE OXIDASE GENES

木质素是世界上第二丰富的可再生有机聚合物。 生物圈。木质素的生物降解是最重要的 地球的碳循环不仅是因为它的丰富 但也是因为它的顽固。裸毛类 黄孢子菌是一种丝状木质素分解担子菌，能产生 H202产生葡萄糖氧化酶及依赖于H202 细胞外的血红素过氧化物酶称为木质素酶，但仅在 特发性酶(次生代谢)。木质素酶构成了一类 六种或更多相关蛋白质，似乎由 不同的基因。最近有很多人对 木质素酶，因为这些蛋白质除了参与 在木质素的降解中，也起着关键的解毒作用 顽固的环境污染物，如二恶英、滴滴涕、 林丹和苯并吡喃，它们构成了严重的公共卫生 危险。我们的长期目标是分离和鉴定 黄孢霉不同木质素降解基因及其研究 它们的分子组织、表达和调控 基因。在这项研究中，我们建议对木质素酶进行表征。 基因组克隆包括构建详细的限制性内切酶 图谱，决定内含子的存在和性质， 基因边界和DNA序列的确定 5‘上游区(约1kb)的测定和 整个编码区。上游监管区域将是 通过DNase I保护分析进行鉴定。转录 如果存在特定的结合蛋白，将被分离并 特色化的。此外，还研究了P. 黄孢子菌及其木质素酶基因在土壤中的分布 染色体(S)将被确定。在这项研究中，我们还建议 葡萄糖基因和基因组克隆的分离与鉴定 另一种在次生代谢中表达的酶--氧化酶 据报道与木质素降解有关 系统。葡萄糖氧化酶的cDNA和基因组克隆将是 其特征与木质素酶克隆的建议相同。 拟议的研究结果不仅将有助于 对P.P.基因结构和组织的理解 黄孢子菌也将成为一个重要的基础 对关于生物和遗传学的知识库的贡献 丝状真菌。所获得的数据还将对 生产基因工程黄孢原毛杆菌菌株 使木质素酶产量高，这是一种具有许多重要作用的酶 实际应用。

Lignin is the second most abundant renewable organic polymer in the biosphere. Lignin biodegradation is of utmost importance in the earth's carbon cycle not only because of its great abundance but also because of its recalcitrance. Phanerochaete chrysosporium, a filamentous ligninolytic basidiomycete, produces H202-generating glucose oxidase and H202 dependent extracellular heme peroxidases called ligninases, but only during idioptase (secondary metabolism). Ligninases constitute a class of six or more related proteins that appear to be encoded by different genes. There has been much recent interest in ligninases because these proteins, in addition to their involvement in degradation of lignin, also play a key role in the detoxification of recalcitrant environmental pollutants such as dioxins, DDT, lindane and benzopyrenes, which constitute a serious public health hazard. Our long term objective is to isolate and characterize various lignin degradation genes from P. chrysosporium and study the molecular organization, expression and regulation of these genes. In this study, we propose to characterize ligninase genomic clones including construction of detailed restriction maps, determine the presence and nature of introns, determination of gene boundaries, and DNA sequence determination of the 5' upstream region (approximately 1 kb) and the entire coding region. Upstream regulatory regions will be identified using DNase I protection analysis. Transcription specific binding proteins, if any present, will be isolated and characterized. Furthermore, chromosome organization of P. chrysosporium and distribution of ligninase genes in the chromosome(s) will be determined. In this study, we also propose to isolate and characterize cDNA and genomic clones for glucose oxidase, another enzyme expressed during secondary metabolism and is reported to be associated with the lignin degradation system. The cDNA and genomic clones for glucose oxidase will be characterized the same way as proposed for the ligninase clones. The results of the proposed research will not only aid in the understanding of gene structure and organization in P. chrysosporium but also will make an important fundamental contribution to the knowledge base on the biology and genetics of filamentous fungi. The data obtained will additionally be useful in producing genetically engineered strains of P. chrysosporium that give high yields of ligninase, an enzyme with many important practical applications.