Characterization And Chemical Modification Of Lipase From Rhizopus ZM-10

The application of lipase has great significance in the wine industry, the Rhizopus in Daqu is one of the main micro-organisms in the formation of the wine aroma components during liquor fermentation process. Luzhou-flavor liquor style and the advantages and disadvantages of wine quality depend on the level of ethyl esters in the wine contents as well as the co-ordination with other substances. Rhizopus lipase is applied in the wine production, but its enzymatic properties and catalytic mechanism is not elucidate and there are many practical problems, such as low enzyme activity, unstable effect and unwide usage and so on. Therefore, the in-depth study and clarify the Rhizopus lipase enzyme nature and role of mechanisms for the application in the food industry have greater theoretical and practical significance. In this paper, the conditions of Rhizopus ZM-10 Lipase purification, the basic enzymatic properties and active amino acid type were studyed. Primarily study results as follows:1. Isolation and purification condition of the Lipase from Rhizopus sp ZM-10: A lipase from Rhizopus sp ZM-10 was using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-100 gel filtration chromatography. This purification protocol resulted in a 15.3-fold purification of lipase with22.2% final yield. The purification conditions: ammonium sulphate saturation 70%; The best conditions of DEAE-Sepharose Fast Flow anion exchange chromatography: sample volume 5mL, elution flow rate 3mL/L, collecting volume 3mL, elution volume 10 times of column volume, eluting salt concentration 0.4mol/L; The best conditions of Sephadex G-100 gel filtration chromatography: sample volume 3mL, elution flow rate easy-flow-rate, collecting volume 1.8mL, elution volume 2 times of column volume The molecular weight of the lipase is approximately 44kDa by SDS-PAGE gel electrophoresis.2. Research on the basic enzymatic properties of the Lipase from Rhizopus sp ZM-10: the research to this purification protocol showed that the optimum pH and temperature for lipolytic activity of the lipase was 8.0and 35℃, respectively. It was extremely stable at 20℃and retained 90% of its original activity for 1h. The lipase was highly stable in the pH range from 7.5 to 8.0 for 4h. Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺and EDTA affected lipolytic activity on the small side, whereas Zn²⁺ ions caused strong inhibition. The organic solvent of various alcohols inhibited lipolytic activity to some extent; the lipase had good stability in the organic solvent of alkanes.3. Chemical modification of the Lipase active sites from Rhizopus sp ZM-10 : Six modifiers (NBS,EDC,DEPC,CH-T,PMSF,DTNB)were used to react with Rhizopus sp ZM-10 lipase.The result indicated that aspartate (glutamate), histidine,serine and tryptophan residues were essential groups for the catalytic activity; aspartate(glutamate), histidine and serine residue were in the substrate binding site,tryptophan residues was important in maintaining the enzyme activity, but not in the substrate binding site of the enzyme.