Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources

- BN-PAGE survey of C. byssicola secretome revealed high molecular protein bands.
- Zymography of BN-PAGE gels displayed xylanolytic and cellulolytic activities on those bands.
- Bands could be decomposed into smaller components indicating they are putative protein complexes.
- LC-MS/MS identified the bands as multienzymatic complexes composed mainly of holocellulases.
- The composition of the putative complexes varied according to lignocellulose carbon source.

Multienzymatic complexes with plant lignocellulose-degrading activities have recently been identified in filamentous fungi secretomes. Such complexes have potential biotechnological applications in the degradation of agro-industrial residues. Fungal species from the Clonostachys genus have been intensively investigated as biocontrol agents; however so far their use as producers of lignocellulose-degrading enzymes has not been extensively explored. Secretomes of Clonostachys byssicola following growth on different carbon sources (passion fruit peel, soybean hulls, cotton gin trash, banana stalk, sugarcane bagasse, orange peel, and a composition of soybean hulls: cotton gin trash:orange peel) were subjected to enzymatic assays. Remarkable differences were observed among the samples, especially regarding levels of mannanase and pectinase activities. Secretomes were then subjected to Blue Native PAGE in order to resolve putative protein complexes which subsequently had their composition revealed by trypsin digestion followed by LC-MS/MS analysis. The protein bands (named I, II, III and IV) were shown to be composed by holocellulolytic enzymes, mainly cellulases and xylanases as well as proteins involved in biocontrol processes, such as chitinases and proteases. The high diversity of proteins found in these multicatalytic assemblies confirms C. byssicola as a novel source of plant biomass-degrading enzymes.

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