Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus.
Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus.
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DOI：
10.1128/aem.00304-16
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发表时间：
2016-08-15
影响因子：
4.4
通讯作者：
Faulds CB
中科院分区：
文献类型：
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作者：
Daou M;Piumi F;Cullen D;Record E;Faulds CB
白色腐烂真菌Pycnoporus cinnabarinus的基因组包括大量编码与木质素降解有关的酶的基因。其中，三个基因被预测为编码乙二醛氧化酶，一种以前从黄孢原毛革菌分离的酶。黄孢原毛平革菌（P. chrysosporium）的乙二醛氧化酶通过产生胞外H2 O2与木质素氧化过氧化物酶生理偶联，并利用一系列醛和α-羟基羰基化合物作为底物。在尼日尔曲霉菌株D15#26（pyrG阴性）中异源产生了预测的朱砂黑曲霉的两种乙二醛氧化酶GLOX 1（PciGLOX 1）和GLOX 2（PciGLOX 2），并使用固定化金属离子亲和色谱法纯化，分别产生59和5 mg的PciGLOX 1和PciGLOX 2蛋白。这两种蛋白质的大小约为60 kDa，并且是N-糖基化的。这些酶的最适活性温度为50°C，最适pH为6。在50°C下孵育4小时后，酶保留其大部分活性。以乙醛酸为底物时，两种酶的相对活性和催化效率最高。这两种朱砂叶甲酶通常表现出相似的底物偏好，但PciGLOX 2显示出更广泛的底物特异性，并且对3-苯基丙醛的活性显著更高。 重要性这项研究解决了真菌过氧化物酶如何获得原位供应的过氧化氢，使他们能够氧化各种有机和无机化合物的作用知之甚少。这种协同活性是生物体内在的，以控制其环境中有毒H2 O2的量，从而提供按需补料的情况，并且可以在生物技术上用于为过氧化物酶反应提供廉价的过氧化物源。作为木质纤维素分解机制的一部分，丝状真菌分泌多种乙二醛氧化酶表明了一种受控系统，特别是当这些酶利用真菌代谢产物作为底物时。两个乙二醛氧化酶已被分离和表征的日期，和由Pycnoporus cinnabarinus产生的两种酶的底物特异性的差异说明了存在于一个单一的真菌，以及利用这些酶来制备平台化学品的工业中的替代机制。
The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.