Food Biotechnology Laboratory, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
J Biotechnol. 2012 Jan 20;157(2):304-14. doi: 10.1016/j.jbiotec.2011.11.021. Epub 2011 Dec 9.
Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research.
尽管子囊菌来源的漆酶在催化特性方面表现出有趣的性质，但已报道和充分表征的数量远小于担子菌来源的漆酶，因此人们对担子菌来源的漆酶的催化机制和工业应用进行了深入研究。我们报告了一种由植物病原菌子囊菌 Botrytis aclada 产生的高度耐受氯化物的漆酶，该漆酶通过毕赤酵母表达系统以极高的产量进行重组表达并纯化至均一性。在分批补料发酵中，在培养基中检测到 495mg/L 的漆酶，这是酵母表达系统获得的漆酶的最高浓度。重组 B. aclada 漆酶的氨基酸链的典型分子量为 61565Da。等电点约为 2.4，对于漆酶来说是一个非常低的值。与重组蛋白结合的糖基残基约占总蛋白质量的 27%。B. aclada 漆酶在酸性和近中性 pH 值下对酚类和非酚类底物表现出非常低的 K（M）值和高底物转化数。在氯离子存在下，酶的稳定性增加，更重要的是，其底物转化仅被氯离子弱抑制（I（50）=1.4M），这与大多数其他描述的漆酶形成鲜明对比。这种高氯容忍度对于某些应用是必需的，例如可植入生物燃料电池和漆酶催化反应，这些应用会受到氯离子的影响。高表达产量允许快速轻松地生产，以进行进一步的基础和应用研究。
