Purification and characterizations of a novel extracellular protease from Shewanella putrefaciens isolated from bigeye tuna

Abstract

Shewanella putrefaciens is one of the common spoilage microorganisms in aquatic products, and its extracellular protease is the main cause of seafood spoilage. The characteristics of the extracellular protease produced by S. putrefaciens YZ08 and its hydrolytic properties on fish proteins were studied. After purification, an extracellular protease of 18.78 kDa was obtained and identified as a metalloprotease of the M23 family. The gene encoding this enzyme was cloned and purified by expression in E. coli to obtain a recombinant protease. YZ08 protease showed optimum enzymatic activity at 40 °C and pH 9.0 and was stable at pH 7.0–10.0 and low temperature (<40 °C). The kinetic parameters and thermal inactivation parameters of the enzyme were determined. S. putrefaciens YZ08 protease can degrade fish myofibrillar and sarcoplasmic proteins, breaking them down into small particles and low molecular peptides. The three-dimensional structure of the protease was simulated by homology modeling and revealed a correlation between the active site in the protein and substrate specificity. This work provides a foundation for characterizing the properties of S. putrefaciens YZ08 protease and for controlling the quality and safety of seafood.