| Lipase is a kind of hydrolase that hydrolyze oils and fats at oil/water interface. Lipase is an important biocatalyst that is widely used in industrial process. Partial glyceride lipase (PGL) is a kind of lipase that act on diacylglyceride or monoacylglyceride, but not triglyceride. PGL is addressed to have potential in industrial application. While in lipase research field, researches about partial glyceride lipase is relatively backward.Lipase SMG1 is a kind of partial glyceride lipase isolated from Malassezia globosa recently, which can hydrolyze mono-and diacylglyceride. The crystall structure of SMG1 in closed form has been resolved (PDB ID:3UUE and 3UUF), which has made the research into structure-function relationship of SMG1 possible. As structure shows, SMG1 contains a lid region in loop conformation, differ from most lipases with a lid region in helix conformation. Lid region in lipase is reported to affect substrate specificity, and its movement from "close" to "open" form in catalyze can lead to a specific phenomenon called "interfacial activation".This research is aimed to study the substrate recognition mechanism of SMG1, getting further information on structure-function relationship of SMG1, and mainly include the following contents:1. Mutants designing. We analyzed crystall structure of SMG1 and found that Phe278 and Leu103 may contribute to the substrate specificity. Phe104 can affect the mobility of lid region. Mutants L103G, F104G, F278A, L103G-F278A, F104G-F278A and Delete(103-104) were designed to study the substrate recognize mechanism of SMG1.2. Preparation of pure enzyme of wild type and mutants. We constructed the expression host of mutants from using site-directed mutation technology, and expressed them in P. X-33. The supernate were consentrated and pured through metal chelating chromatography.3. Biochemical properties analysis and comparision of wild type and mutants.1) Fistly, we characterized the interfical catalyzing character of SMG1-Wt using p-nitrophenol butyrate (PNPB) as substrate. Results showed that SMG1 does not show a typical activation curve as most lipase do, we suppose it may connect to the special lid region of SMG1.2) Kinetic constant and substrate specificity of these mutants and wild type were measured. Results showed that all mutants show no activity towards TAG, while preference toward 1,3-DAG of mutants L103G-F278A, F104G-F278A and Delete(103-104) has been largely improved. The ratio of 1,3-DAG hydrolyzing rate to 1,2-DAG of L103G-F278A, F104G-F278A and Delete(103-104) calculated to be 7.21, 10.17 and 6.42 respectively, compared to 1.38 of wild type.3) Specific activity to artificial substrate (PNP esters) and DAG of these mutants has also been analyzed. F278A retained about 80% activity toward DAG emulsion while only 11% activity toward PNPO. L103G increased its activity upon PNPO by about 2 folds while F104G showed about 40% decrease in PNPO activity, and they both showed decreased activity upon DAG emulsion (53% and 35% respectively). Delele103-104 retained about 30% activity toward DAG emulsion with almost completely loss of PNPO activity.4)Based on the modulation of specificity and activity observed, a PNPO binding model for ester (Asn102 and Phe278 forming flexible bridge) and specific lipid-anchoring mechanism for DAG (Leu 103 and Phe104 serving as "anchors" to lipid interface) were proposed.4. Research into the relationship between Asn277 and thermostability of SMG1. Mutants N277D, N277F, N277V, N277L were constructed by lab members before. Circular dichroism spectra were used to analyze the thermal stability of Asn277 mutants. Results were in consistent with the data measured before. Molecular modeling of mutants were constructed to understanding the mechanism of effect to thermal stability of Asn277 to SMG1. |
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