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首页> 外文期刊>Applied Microbiology and Biotechnology >Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene
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Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

机译：水生真菌Myrioconium sp UHH 1-13-18-4漆酶的纯化，生化特性及漆酶编码基因的分子分析

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摘要

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flightmass spectrometry and an isoelectric point below 2.8 was purified from CuSO4 and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k(cat)/K-m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzthiazoline6- sulfonate) were 67.3, 46.9, and 28.2 s(-1) mM(-1), respectively, with Km values of 4.2, 67.8, and 104.9 mu M. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15 C. The laccase encoding gene was identified and sequenced at both the genomic and complementary DNA ( cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.
机译：Myrioconium sp。 UHH 1-13-18-4菌株是从德国中部萨勒河中分离出来的一种子囊无脊椎动物。通过基质辅助激光解吸/电离飞行时间质谱法测定的等离子分子量为72.7 kDa的细胞外，单体和糖基化的漆酶，从CuSO4和在麦芽中生长的香草酸修饰的液体真菌培养物中纯化得到，其等电点低于2.8。提取培养基。丁香嗪，2,6-二甲氧基苯酚和2,2'-叠氮基双（3-乙基苯并噻唑啉6-磺酸盐）的氧化催化效率（k（cat）/ Km）为67.3、46.9和28.2 s（-1 ）mM（-1），Km值分别为4.2、67.8和104.9μM。在不同pH值和温度下预孵育1 h后，pH 4保留了80％的初始漆酶活性。鉴定到漆酶编码基因并在基因组和互补DNA（cDNA）水平上测序，并报道了启动子区域的相应结构特征和假定的调控元件。通过质谱鉴定纯化的酶的两个胰蛋白酶肽证实了功能漆酶蛋白与Myrioconium sp的翻译的基因组序列相同。漆酶。 Myrioconium sp。漆酶与来自巩膜菌科（Sclerotiniaceae）的子囊菌（Helicolias）的子囊菌的漆酶显示出最高的同一性。

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