Analysis of biopulping mechanism by Ceriporiopsis subvermispora

枯草芽孢杆菌(Ceriporiopsis Subvermispora)是一种特殊的木质素降解真菌，近年来已被用于生物机械制浆、生物亚硫酸盐制浆和提高牧草消化率等植物生物质的脱木素处理。已知这种真菌分解位于木材细胞壁和中间薄片中的木质素，即使在纤维分离后也不会侵蚀木材细胞壁。考虑到菌丝生长和酶的分泌是在管腔内进行的，而且酶太大而不能穿透木材细胞壁孔，因此可以推断，真菌产生了某种可扩散的低分子化合物，导致木质素在远离酶的位置降解。在枯草芽孢杆菌生物机械法制浆中，具有较高的节能效果，潜伏期较短，仅为2周，说明中板在前2周就受到了破坏。培养6周后，体重相应下降15-20%，…显微镜下可观察到更多的纤维通过中层板层的木质分解而分离。因此，枯草芽孢杆菌对木材腐烂早期中层板层的广泛破坏不能用简单的从细胞腔到壁区的自由基转移系统来理解。在此基础上，我们重点研究了木材细胞壁内的自由基产生系统。本研究对枯草芽孢杆菌产生的小分子过氧化产物进行了GC-MS、核磁共振和电子自旋共振波谱分析。结果发现，枯草芽孢杆菌在木粉培养上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)十八酸和十六酸。用大豆脂肪氧合酶氧化含有USFA和SFA的组分，用4-POBN自旋捕集分析酶促过氧化生成的自由基。ESR谱表明，在培养早期(8-12天)得到的提取物产生了强烈的自由基。然而，长时间培养(16-28天)后，自旋加合物的信号强度并没有增加。主要自旋加合物的超精细耦合常数和g值与戊基自由基相同。这些结果有力地表明，游离不饱和脂肪酸不是来自真菌菌丝，而是与木质素生物降解有关的真菌胞外代谢产物。DPPP实验也证明该菌能产生脂质过氧化氢。较少

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