Cassland P, Jönsson L J
Lund University/Lund Institute of Technology, Sweden.
Appl Microbiol Biotechnol. 1999 Sep;52(3):393-400. doi: 10.1007/s002530051537.
Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 degrees C instead of 28 degrees C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance.
漆酶可用于木质纤维素水解产物的酶解毒。通过漆酶的异源表达，可能会获得一种对酚类抑制剂具有增强抗性从而提高发酵木质纤维素水解产物能力的酿酒酵母菌株。对来自木质素降解担子菌云芝的新型漆酶基因lcc2的cDNA进行测序表明，它编码一种由499个氨基酸残基组成的同工酶，前面有一个21个残基的信号肽。通过与埃德曼降解数据比较，得出lcc2编码与漆酶A相对应的同工酶的结论。lcc2的基因产物与先前鉴定的云芝lcc1基因产物具有71%的同一性。漆酶序列比对显示，云芝同工酶一般与其他白腐真菌的相应同工酶比与云芝的其他同工酶关系更密切。因此，漆酶的多样性是云芝和相关白腐真菌物种的一个保守特征。当云芝lcc2 cDNA在酿酒酵母中表达时，活性酶的产生强烈依赖于温度。培养3天后，阳性转化体在19℃而非28℃保存时，漆酶活性高出16倍。用表达云芝漆酶基因lcc1的毕赤酵母进行的类似实验也表明，较低的培养温度对表达水平有很大的促进作用，这表明在酿酒酵母表达系统中观察到的现象具有普遍意义。
