Pseudomonas ilişkili genuslarda ekstraselüler lipaz aktivitesi ve Stenothrophomonas maltophilia MU 53 suşundan lipaz saflaştırılması

Abstract

VI EXTRACELLULAR LIPASE ACTIVITY OF PSEUDOMONAS RELATED GENERA AND PURIFICATION OF LIPASE FROM Stenothrophomonas maltophilia MU 53 (M.Sc. Thesis ) Havser ERTEM MUGLA UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY 2005 ABSTRACT In this study, eighteen different strains of fish isolates, eleven belonging to Stenotrophomonas maltophilia, five belonging to Chryseomonas luteola and two belonging to Sphingomonas paucimobilis were tested for their qualitative esterase activity and extracellular lipase production. Quantitative extracellular lipase activity was also estimated through spectrophotometric assay and it ranged between 0.312- 2.534 U/ml. The lipase production was substantially enhanced when different types of carbon and nitrogen sources are added to culture medium (NBCM3). ForVII optimization of lipase production, a variety of oils, such as olive oil, sunflower oil, soybean oil, almond oil, hazelnut oil, tributyrin, fish oil were added to culture medium as a carbon source (1.5 %, v/v) and sodium nitrate, ammonium sulfate, ammonium nitrate, ammonium chloride and urea (1.5% v/v) were added to culture media as a nitrogen source. While fifteen strains showed maximum activity when carbon source was sunflower oil, two strains showed maximum activity when carbon source was soybean oil and one strain showed maximum activity when carbon source was olive oil in the culture medium. All eighteen strains showed maximum activity when nitrogen source was ammonium sulfate in the culture medium. S. maltophilia MU 53 was the highest lipase-producing strain with the activity level of 2.534 U/ml. The maximum lipase production of this strain was detected in the culture medium containing sunflower oil as a carbon source and ammonium sulfate as a nitrogen source. The extracellular lipase of S.maltophilia MU 53 was homogeneously purified using a combination of ammonium sulfate precipitation, dialyze and gel filtration column chromatography with Sephadex G-100. The total protein and lipase activity of fractions that isolated through purification steps were analyzed by Bradford assay and spectrophotometric assay, respectively. The enzyme was purified 25.95 fold with a yield of 39.8 % and specific activity of 27.69 U/mg protein. Protein samples collected at the purification steps were extracted and, then, they were separated according to their molecular weights with SDS-PAGE technique. All the protein samples collected were run on the same gel for comparisons of protein bands. The purified extracellular lipase showed one protein band in SDS-PAGE with molecular weight 87.96 kDa. Key Words: Lipase, Stenotrophomonas maltophilia, Chryseomonas luteola, Sphingomonas paucimobilis, purification Page number : 86 Adviser : Doç.Dr. Aysel UĞUR

VI EXTRACELLULAR LIPASE ACTIVITY OF PSEUDOMONAS RELATED GENERA AND PURIFICATION OF LIPASE FROM Stenothrophomonas maltophilia MU 53 (M.Sc. Thesis ) Havser ERTEM MUGLA UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY 2005 ABSTRACT In this study, eighteen different strains of fish isolates, eleven belonging to Stenotrophomonas maltophilia, five belonging to Chryseomonas luteola and two belonging to Sphingomonas paucimobilis were tested for their qualitative esterase activity and extracellular lipase production. Quantitative extracellular lipase activity was also estimated through spectrophotometric assay and it ranged between 0.312- 2.534 U/ml. The lipase production was substantially enhanced when different types of carbon and nitrogen sources are added to culture medium (NBCM3). ForVII optimization of lipase production, a variety of oils, such as olive oil, sunflower oil, soybean oil, almond oil, hazelnut oil, tributyrin, fish oil were added to culture medium as a carbon source (1.5 %, v/v) and sodium nitrate, ammonium sulfate, ammonium nitrate, ammonium chloride and urea (1.5% v/v) were added to culture media as a nitrogen source. While fifteen strains showed maximum activity when carbon source was sunflower oil, two strains showed maximum activity when carbon source was soybean oil and one strain showed maximum activity when carbon source was olive oil in the culture medium. All eighteen strains showed maximum activity when nitrogen source was ammonium sulfate in the culture medium. S. maltophilia MU 53 was the highest lipase-producing strain with the activity level of 2.534 U/ml. The maximum lipase production of this strain was detected in the culture medium containing sunflower oil as a carbon source and ammonium sulfate as a nitrogen source. The extracellular lipase of S.maltophilia MU 53 was homogeneously purified using a combination of ammonium sulfate precipitation, dialyze and gel filtration column chromatography with Sephadex G-100. The total protein and lipase activity of fractions that isolated through purification steps were analyzed by Bradford assay and spectrophotometric assay, respectively. The enzyme was purified 25.95 fold with a yield of 39.8 % and specific activity of 27.69 U/mg protein. Protein samples collected at the purification steps were extracted and, then, they were separated according to their molecular weights with SDS-PAGE technique. All the protein samples collected were run on the same gel for comparisons of protein bands. The purified extracellular lipase showed one protein band in SDS-PAGE with molecular weight 87.96 kDa. Key Words: Lipase, Stenotrophomonas maltophilia, Chryseomonas luteola, Sphingomonas paucimobilis, purification Page number : 86 Adviser : Doç.Dr. Aysel U?UR