Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Lyngby, Denmark.
Enzyme Microb Technol. 2013 Dec 10;53(6-7):378-85. doi: 10.1016/j.enzmictec.2013.08.003. Epub 2013 Aug 28.
Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin. When grown on malt extract or minimal medium supplemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited high laccase activity, ∼3U/mL toward syringaldazine. This activity was 13-17 fold higher than the corresponding activities of the crude protein extracts of P. brumalis, P. ciliatus and T. versicolor. Native PAGE electrophoresis of the crude G. lucidum extract confirmed the presence of an active laccase. The G. lucidum laccase had a molecular weight of ∼62.5kDa, and a Km value of 0.107mM (determined on ABTS). A partial amino acid sequence analysis of four short de novo sequenced peptides, defined after trypsin digest analysis using MALDI-TOF MS/MS analysis, revealed 64-100% homology to sequences in related laccases in the UniProt database, but also indicated that certain sequence stretches had low homology. Addition of the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) together with a state-of-the-art cellulase enzyme preparation (Cellic™CTec1) produced significantly increased cellulolytic yields, which were also better than those obtained with a T. versicolor laccase addition, indicating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosic biomass conversion.
基于对补充木质纤维素的最小培养基上的 44 种白腐真菌进行差异预筛选，选择了四种担子菌进行进一步研究：灵芝、猪苓、糙皮侧耳和彩绒革盖菌。只有灵芝能够在麦芽提取物或补充碱木质素的最小培养基上生动生长。当在补充木质纤维素（甘蔗渣）的麦芽提取物或最小培养基上生长时，粗灵芝蛋白提取物表现出高漆酶活性，对邻苯二胺的活性约为 3U/mL。该活性比猪苓、糙皮侧耳和彩绒革盖菌粗蛋白提取物的相应活性高 13-17 倍。粗灵芝提取物的 native PAGE 电泳证实存在活性漆酶。灵芝漆酶的分子量约为 62.5kDa，Km 值为 0.107mM（在 ABTS 上测定）。通过 MALDI-TOF MS/MS 分析对四个短从头测序肽进行胰蛋白酶消化分析后定义的肽，与 UniProt 数据库中相关漆酶的序列具有 64-100%的同源性，但也表明某些序列片段具有低同源性。将富含漆酶的灵芝菌液添加到木质纤维素生物质（预处理的甘蔗渣）中，再加上最先进的纤维素酶制剂（Cellic™CTec1），可显著提高纤维素酶的产率，甚至优于添加彩绒革盖菌漆酶的产率，这表明灵芝漆酶具有独特的特性，可能在木质纤维素生物质转化中具有重要意义。
