Balcázar-López Edgar, Méndez-Lorenzo Luz Helena, Batista-García Ramón Alberto, Esquivel-Naranjo Ulises, Ayala Marcela, Kumar Vaidyanathan Vinoth, Savary Olivier, Cabana Hubert, Herrera-Estrella Alfredo, Folch-Mallol Jorge Luis
Facultad de Ciencias, Universidad Autónoma del estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Mor, México.
Centro de Investigación en Biotecnología, Universidad Autónoma del estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, México.
PLoS One. 2016 Feb 5;11(2):e0147997. doi: 10.1371/journal.pone.0147997. eCollection 2016.
Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.
真菌漆酶是一类因其能够使废水脱色和解毒而被研究的酶；它们还被用于纸张漂白、聚合物合成、生物修复等领域。在这项工作中，我们成功地在丝状真菌深绿木霉中表达了血红栓菌（红孔菌）的漆酶。为此，设计了一个转化载体，将目的基因整合到靠近blu17终止子区域的基因间位点。尽管仍然需要单孢子筛选，但在所需位点实现了稳定整合。血红栓菌漆酶的天然信号肽成功地将重组蛋白分泌到培养基中。纯化后的异源表达漆酶保持了与天然酶相似的特性（ABTS的Km、kcat和kcat/km值、热稳定性、底物范围、最适pH等）。为了确定这种改良菌株的生物修复潜力，使用过表达漆酶的木霉菌株去除外源化合物。与野生型相比，这种改良菌株能更有效地去除工业废水中存在的酚类化合物和培养基中的双酚A（一种内分泌干扰物）。此外，异源表达的漆酶能够使不同的染料脱色，并在体外去除苯并[a]芘和菲，显示出其对外源化合物的降解潜力。
