Is Fungal Laccase Truly Necessary for Lignin Degradation?

酚氧化酶是白腐菌降解木质素的必需酶。已知的在这一过程中起作用的酚氧化酶是木质素过氧化物酶(LIP)、锰过氧化物酶(MNP)或漆酶。黄孢原毛平革菌是研究木质素降解酶机制的模式生物，产生多种形式的LIP和MNP，但漆酶不作为木质素分解系统的一部分。现在越来越明显的是，大多数白腐菌与一组不同的酚氧化酶一起工作，即漆酶与LIP或MNP或甚至三种酶一起工作。为了研究漆酶在木质素降解中的作用，朱砂拟青霉被确定为一种优良的生物，因为它只产生一种等电型的漆酶。为了研究漆酶的重要性，将使用产生和鉴定突变体的经典方法，即无漆酶(lac-)和无过氧化物酶(per-)，以及多效性(即lac-per-)突变体。还将在漆酶的物理化学和光谱表征方面花费大量的努力。本实验室早先开发的用于克隆植物漆酶基因的材料和程序将用于克隆编码朱砂假单胞菌漆酶的基因组DNA序列。这项研究将提供关于真菌漆酶在木质素降解中的作用的信息，并与研究得很好的黄孢原毛平革菌系统进行比较。20世纪70年代的石油危机使人们的兴趣转向可再生资源的利用，特别是木质纤维素。木质纤维材料是纤维、能源和液体燃料的来源。然而，目前利用这种原材料的技术大多是耗能的，而且并不总是环境友好的。由于自然界最重要的生物反应之一是木材和其他木质纤维材料的降解，因此开发利用来自农业和林业的木质纤维材料的生物技术方法是可能的，也是重要的。在这种情况下，降解木质素的有效方法尤为重要。白腐菌是唯一能在一定程度上降解木质素的微生物。要有效地利用这些微生物更好地利用木质纤维材料，需要对它们在木质素降解过程中利用的酶机制有深入的了解。我们将在拟议的研究中采用的方法将特别提供关于漆酶的作用的信息，即漆酶是否是木质素降解所必需的酚氧化酶之一。

Phenoloxidases are absolutely essential for lignin degradation by white-rot fungi. The known phenoloxidases functioning in the process are lignin peroxidase (LiP), manganese peroxidase (MnP), or laccase. Phanerochaete chrysosporium, the model organism for studies of the enzyme mechanisms involved in lignin degradation, produces multiple forms of both LiP and MnP, but laccase is not used as part of this ligninolytic system. It is now becoming evident that the majority of white-rot fungi operate with a different set of phenoloxidases, i.e., laccase in combination with either LiP or MnP or even all three together. To investigate the role of laccase for lignin degradation Pycnoporus cinnabarinus was identified as an excellent organism for further study because it produced only one isoelectric form of laccase. To investigate the importance of laccase, the classical approaches of creating and identifying mutants, i.e., laccase-less (lac-) and peroxidase-less (per-), as well as pleiotropic (i.e., lac-per-), mutants will be used. Substantial efforts will also be expended for the physico-chemical and spectroscopic characterization of the laccase. Materials and procedures earlier developed in this laboratory for cloning of plant laccase genes, will be used to clone the genomic DNA sequence encoding the P. cinnabarinus laccase. This research will provide information on the role of fungal laccases in lignin degradation to be compared with that of the well studied P. chrysosporium system. %%% The oil crisis during the 1970's turned interest towards the utilization of renewable resources, and towards lignocellulosics, in particular. Lignocellulosic materials are a source of fiber, energy and liquid fuels. However, the technologies presently used to take advantage of this raw material are mostly energy consuming and not always environmentally friendly. Since one of nature's most important biological reactions is the degradation of wood and other lignocellulosic material, biotechnical methods for utilization of lignocellulosic materials from agriculture and forestry are both possible and important to develop. Efficient ways to degrade lignin are particularly important in this context. White-rot fungi are the only microorganisms which can degrade lignin to any extent. To efficiently use these organisms for a better utilization of lignocellulosic materials requires an incisive knowledge of the enzyme mechanisms they utilize in the lignin degradation process. The approach we will take in our proposed research will particularly provide information on the role of laccase, i.e., whether or not this is one of the necessary phenoloxidases for lignin degradation.