Lauber Christiane, Schwarz Tatiana, Nguyen Quoc Khanh, Lorenz Patrick, Lochnit Guenter, Zorn Holger
Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Heinrich-Buff-Ring 17, 35392, Giessen, Germany.
AB Enzymes GmbH, Feldbergstrasse 78, 64293, Darmstadt, Germany.
AMB Express. 2017 Aug 23;7(1):164. doi: 10.1186/s13568-017-0463-5.
The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6-dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of HO by rPsaDyP, however enzyme activity was increased by the addition of HO. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.
从美味侧耳菌分泌组中克隆出一种过氧化物酶的编码序列，该序列来自一个cDNA文库。生物信息学分析显示，其开放阅读框为1551 bp，对应一个由516个氨基酸组成的初级翻译产物。该DyP型过氧化物酶在里氏木霉中进行了异源表达，活性为55,000 U/L。从培养上清液中纯化该酶，对其进行生化特性分析并测定动力学参数。该酶具有一个由62个氨基酸组成的N端信号肽。通过蓝色非变性聚丙烯酰胺凝胶电泳和ABTS活性染色分析以及凝胶过滤色谱显示该酶的天然二聚体状态（115 kDa）。底物范围分析表明，除了催化一些经典过氧化物酶底物的转化，如2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)和取代酚类如2,6-二甲氧基苯酚外，该重组酶还能催化蒽醌染料活性蓝5的脱色。该酶还能催化天然色素如β-胡萝卜素和胭脂树橙的脱色。令人惊讶的是，无论有无H₂O₂，rPsaDyP都能转化β-胡萝卜素，不过添加H₂O₂会提高酶活性。这表明rPsaDyP除了具有过氧化物酶活性外还具有氧化酶功能。由于对特征底物活性蓝5具有高亲和力，rPsaDyP在功能上属于dyp型过氧化物酶家族。
