Amino acid sequence of two new milk-clotting proteases from the macroalga Gracilaria edulis

This study is aimed at identifying and characterising the proteases we previously extracted from the red seaweed Gracilaria edulis with the potential as milk-clotting enzymes. The protease extract was first analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Two protease bands with a molecular weight of 44 and 108 kDa were identified, and analysed using in-gel digestion and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS). Eight peptides from the LC-MS/MS analysis matched those in existing protein databases but they were not related to any protease of the genera Gracilaria and Hydropuntia. Further analysis revealed that more than 80% of the peptide sequence of the algal proteases matched with those from members of the bacteria kingdom, including Gallaecimonas and Alteromonas. Among these, twelve matching homolog proteases were identified as metalloprotease and serine protease. The results indicated that the algal proteases have a close relationship with both algae and bacteria, and suggest that the proteases might have resulted from past bacterial colonisation of the algae and subsequent horizontal gene transfer between bacteria and algae.