Purification and Partial Characterization of Psychrotrophic Serratia marcescens Lipase

Abstract

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6h at 30°C and after 12h at 6°C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24h at 4°C in buffer of pH 6.6. The optimum temperature was observed at 37°C and exhibited high activity at 5°C. The thermal inactivation of S. marcescens lipase was more obvious at 80°C; it retained about 15% of its original activity at 80°C and was completely inactivated after heating at 90°C for 5min. Under optimum conditions, activity of the enzyme was maximum after 6min. The Michaelis-Menten constant was 1.35mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5°C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6°C after 12h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.