Lipase was partially purified from the viscera of grey mullet ( Mugil cephalus) by ammonium sulfate fractionation, simultaneous desalting, and concentration via ultrafiltration and then affinity chromatography on EAH-Sepharose 4B. The partially purified extract was characterized using p-nitrophenyl palmitate (rho-NPP) as substrate. Grey mullet lipase was active within the pH range of 7-10, with an optimum pH of 8.0, and was stable from pH 4-10. The enzyme was active within the temperature range of 20°C and 60°C, and exhibited an optimum for the hydrolysis of rho-NPP at 50°C. The enzyme was stable between 10-50°C, beyond which it lost activity progressively. At 50°C there was ca. 50% residual activity after 60 min incubation. However at 60°C, there was 22%, 20% and 0% remaining activity after 10, 30 and 60 min incubation respectively. Based on the temperature activity data, the activation energy for the hydrolysis of rho-NPP was calculated as 1.94 kcal/mol (8.15 kJ/mol). / The rho-nitrophenyl esters of medium to long chain fatty acid (C10-C16) served as good substrates with the order of ease of hydrolysis as; rho-NP-palmitate > rho-NP-myristate > rho-NP-caprate > rho-NP-caproate > rho-NP-butyrate > rho-NP-acetate. The Km' and Vmax for the hydrolysis of rho-NPP were 0.22 mM and 20 mumol min-1 mg-1 , respectively. The hydrolytic activity of the lipase was enhanced by Mg2+, Mn2+, NaN3, and EDTA, but strongly inhibited by Hg2+, and Cu2+. PMSF (1 mM), Ca2+ (1 mM and 10 mM) had no effect on grey mullet lipase activity. Lower concentrations (25-10% v/v) of water-miscible organic solvents (dimethyl sulfoxide, dimethyl formamide, iso-propanol, and methanol) had negligible effect on the activity of the lipase while higher concentrations (>50% v/v) completely inhibited the enzyme. The grey mullet lipase was remarkably stable in water-immiscible organic solvents (benzene, toluene, hexane, heptane, and isooctane). The water-immiscible solvents also activated the enzyme with hexane giving the most activation. Lower concentrations of trihydroxylated bile salts (sodium taurocholate, and sodium cholate) were more potent activators than the dihydroxylated bile salt (sodium deoxycholate). Sodium dodecyl sulfate at 1 mM, and Tween 80RTM at 1% had 6% and 12% stimulatory effect on the activity of the enzyme respectively, while 1% and 0.5% Triton RTM X-100 caused 67% and 40% inhibition, respectively.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.97895 |
| Date | January 2005 |
| Creators | Aryee, Alberta Naa Ayeley. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Food Science and Agricultural Chemistry.) |
| Rights | © Alberta Naa Ayeley Aryee, 2005 |
| Relation | alephsysno: 002479963, proquestno: AAIMR24604, Theses scanned by UMI/ProQuest. |
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