The purification, identification, and characterization of a thermostable metalloprotease in serratia grimesii from channel catfish (ictalurus punctatus) spoilage
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Abstract
Food spoilage and food poisoning in fishery products is an emerging issue affecting the health of consumers throughout the world. Fish are susceptible to various specific spoilage organisms (SSOs) that produce extracellular enzymes during low temperature storage which deteriorates the tissue after fillet processing. In this study, Serratia grimesii was isolated from fillets of channel catfish (Ictalurus punctatus). The protease producing bacteria from catfish fillets where identified through 16S rDNA sequencing and categorized through a phylogenic tree where most isolated SSOs were closely neighbored except S. grimesii. This species was unique from the other protease producing bacteria isolated on the fillet as to its protease protein profile. The goal of this study was to investigate a thermostable metalloprotease from S. grimesii through purification, identification, and characterization. The growth of S. grimesii and its protease production were measured to examine the secretion properties. Optimal growth conditions were achieved at pH 7 and 28 ºC as psychrotrophic bacteria. The protease secretion was best during the log phase of the bacterial growth.
The extracellular crude proteins were isolated after protein induction for three days before biochemical properties were determined by enzyme assays. The crude protein was thermostable and tolerant of a wide range of pH values, solvents, and co-factors. Distinct bands from SDS-PAGE were also observed from the crude protein. The target protein migrated in the gel underneath the 50 kDa was selected because it showed the greatest protease production. The target protease was purified through Next Generation Chromatography using ion exchange and gel filtration columns. Using the ion exchange chromatography, the pH of 8.6 was optimal for found to separate the target protease. The purified protein had a 10-fold increase in specific activity compared to the crude protein preparation. Following purification, biochemical studies were performed to characterize the protease. The protease was thermostable even up to 100 ºC. The activity of the protease was highest at pH 9, however it was still stable in some acidic and alkaline ranges. Manganese was a co-factor that increased activity. Solvents decreased the activity to around 50%. Detergents showed varied results with Triton X-100 not having an effect but SDS inhibiting protease activity. The substrate casein was hydrolyzed the best. To identify the protease, peptides were sequenced using MALDI-TOF-TOF mass spectrometry. The mass spectrometry result of the target enzyme identified as a stable metalloprotease precursor. It is believed that extracellular proteases including this metalloprotease are a major cause of rapid spoilage growth in fish tissue.