Purification and partial characterization of psychrotrophic Serratia marcescens lipase.
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 6 h at 30 degrees C and after 12 h at 6 degrees 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 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM 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 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.
Abdou
Adham M
AM
Department of Food Hygiene, Faculty of Veterinary Medicine, Benha University, Moshtohor 13736 Kaliobyia, Egypt. dradham@yahoo.com
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003611:Dairy Products / Q000032:analysis / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005511:Food Handling / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008892:Milk / Q000382:microbiology*; D008970:Molecular Weight; D012706:Serratia marcescens / Q000201:enzymology*; D013649:Taste; D013696:Temperature; D013997:Time Factors
D008049:Lipase