Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase
Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase
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DOI：
10.1007/s00253-008-1507-1
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发表时间：
2008-07-01
影响因子：
5
通讯作者：
Sato, Toshitsugu
中科院分区：
文献类型：
--
作者：
Sakamoto, Yuichi;Nakade, Keiko;Sato, Toshitsugu
漆酶是由白腐真菌产生的一种木质素降解酶，由于其具有氧化酚类化合物的能力而成为生物修复领域的研究热点。在这份报告中，我们描述了分离和表征的Lcc 1，一个新的基因的香菇编码Lcc 1，并证明重组Lcc 1表达在一个活跃的，分泌的形式在烟草BY-2细胞培养。lcc 1的开放阅读框长1，557个碱基对，编码518个氨基酸的推定蛋白质。我们将嵌合形式的lcc 1（CaMV 35 Sp：clcc 1）导入烟草BY-2细胞，并获得了几个稳定的clcc 1转化体，表达活性Lcc 1。Lcc 1在BY-2培养基中的活性高于在细胞提取物中的活性，这表明重组Lcc 1以分泌形式产生。重组Lcc 1的表观分子量小于从L. edodes。纯化的重组Lcc 1的底物特异性与L. edodes Lcc 1，并且两种酶都能够使同一组染料脱色。这些结果表明，真菌Lcc 1在BY-2细胞中的异源表达将是一个有价值的工具，为生产足够数量的活性漆酶的生物修复。
Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.