Identification of Extracellular Proteases Induced by Nitrogen-Limited Conditions in the Thraustochytrids Schizochytrium aggregatum ATCC 28209

Abstract

Thraustochytrids have attracted attention due to the high contents of useful lipids and growth rate. Genus Schizochytrium is commonly used for docosahexaenoic acid (DHA) production, while a strain, which produces a high amount of squalene, has been reported in the genus Aurantiochytrium. These organisms are heterotrophic, and Schizochytrium degrades the extracellular macromolecules, e.g., proteins and polysaccharides, as the nutrients. However, the extracellular lytic enzymes are not well-studied yet. Here, we investigated the induction of extracellular proteases of Schizochytrium aggregatum ATCC 28209. A casein-hydrolytic activity was induced in the nitrogen-limited conditions, and that was also detected by zymography after fractionation by non-heat denatured SDS-PAGE. The proteinous band corresponding to the protease activity was analyzed by MALDI-TOF mass spectrometry after digestion with trypsin. The molecular mass data of the protein fragments were compared to the protein database of S. aggregatum ATCC 28209 in the Joint Genome Institute, and we found that the molecular masses of the six peptides were matched with the prediction from the sequence of a protein, ID 63992, which was annotated as a peptidase S8 family protein. Interestingly, we found that a paralogous protein, ID 99856, was encoded by a gene flanking at the downstream of the gene for ID 63992, and the expression of both genes was similarly induced under the nitrogen-limited conditions. These findings may provide us a key to disclose the induction mechanisms of the extracellular lytic enzymes and the function of the proteolytic enzyme for the nutrition acquisition in thraustochytrids.

Keywords: Peptidase S8 family; Protein mass fingerprinting; Secretome; Serine peptidases; Subtilisin.