Analysis of biopulping mechanism by Ceriporiopsis subvermispora

近几年来，拟蜡孔菌（Ceriporiopsis subvermispora）作为一种木质素降解真菌，在生物机械法制浆、生物亚硫酸盐法制浆和提高牧草消化率等植物生物质脱木质素处理中得到了广泛的应用。已知这种真菌分解位于木材细胞壁和中层中的木质素，即使在纤维分离后也不侵蚀木材细胞壁。考虑到菌丝生长和酶的分泌发生在管腔中，而酶太大而不能穿透木材细胞壁的孔隙，可以得出结论，真菌产生某种可扩散的低分子量化合物，导致远离酶的部位的木质素降解。在亚蠕孢梭菌生物机械制浆中，培养时间较短，仅2周，说明在前2周中层已被破坏，节能效果较高。培养6周后，对应于15- 20%的重量损失， 关于我们 可以通过显微镜观察到通过中层木质素分解的纤维分离。因此，不能用简单的自由基从细胞腔转移到壁区的系统来理解亚蠕孢梭菌在木材腐朽早期对中层的广泛破坏。在此基础上，重点研究了木材细胞壁内自由基的产生系统。本文采用GC-MS、NMR和ESR等方法对亚蠕孢梭菌产生的可过氧化小分子化合物进行了分析。结果，已经发现，在木粉培养物上，C.subvermispora产生游离不饱和脂肪酸（USFA）、9，12-十八碳二烯酸、11-十八碳烯酸和9-十八碳烯酸，以及饱和脂肪酸（SFA）十八烷酸和十六烷酸。用大豆脂肪氧合酶对含有超饱和脂肪酸和饱和脂肪酸的组分进行氧化，用4-POBN自旋捕集法对氧化产物进行分析，ESR谱表明，在培养初期（8-12 d）提取物中产生了较强的自由基。然而，在长时间培养（16-28天）后，自旋加合物的信号强度没有增加。超精细耦合常数和g值的主要自旋加合物是相同的戊基自由基。这些结果强烈表明，游离不饱和脂肪酸不是来自真菌菌丝，而是来自真菌的胞外代谢产物与木质素的生物降解。DPPP试验也证明了该真菌产生脂质过氧化氢。少

Ceriporiopsis subvermispora, a specific lignin-degrading fungus, has in recent years been used in delignifying treatments of plant biomass such as biomechanical pulping, biosulfite pulping and digestibility enhancement of forage grasses. This fungus is known to decompose lignin located in wood cell walls and middle lamellae without erosion of the wood cell walls even after fiber separation. Taking into account that hyphal growth and excretion of enzymes takes place in the lumen and that enzymes are too large to penetrate pores of wood cell Walls, it can be concluded that some kind of diffusible low molecular weight compounds are produced by the fungus, leading to the degradation of lignin at the site far from enzymes. In the biomechanical pulping with C.subvermispora, high energy saving effect was obtained with a short incubation period of 2 weeks, indicating that middle lamellae were damaged for the first 2 weeks. After 6 weeks cultivation, corresponding to 15-20 percent weight loss, … More fiber separation by the lignolysis of middle lamellae can be observed by microscopy. Thus, extensive damage of middle lamellae in the early stage of wood decay by C.subvermispora cannot be understood by the simple radical transfer system from cell lumina into wall regions. Based on this discussion, we focused on the free radical generation system inside of wood cell walls. In this study, peroxidizable low molecular mass compounds produced by C.subvermispora were analyzed by GC-MS, NMR and ESR spectroscopy. As a result, it has been found that C.subvermispora produced free unsaturated fatty acids (USFA), 9,12- octadecadienoic, 11-octadecenoic and 9-octadecenoic acids, together with saturated fatty acids (SFA) octadecanoic and hexadecanoic acids on wood meal cultures. The fractions containing USFA and SFA were oxidized with soybean lipoxygenase and the free radicals formed by the enzymatic peroxidation were analyzed by spin-trapping with 4-POBN.ESR spectra showed that strong radicals were generated from the extract obtained at the early stages of cultivation (8-12 days). However, signal intensities of the spin adducts did not increase after prolonged cultivation (16-28 days). Hyperfine coupling constant and g-value of the major spin adduct were identical to those of pentyl radical. These results strongly suggest that the free unsaturated fatty acids did not originate from fungal hypha but from extracellular metabolites of the fungus related to lignin biodegradation. Production of lipid hydroperoxide by this fungus was also demonstrated by DPPP assay. Less