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Characterization and Chemical Modification of Lipase from Rhizopus ZM-10

Author: YuZhongLiang
Tutor: ChenYiLun
School: Shandong Agricultural University
Course: Fermentation Engineering
Keywords: Rhizopus Lipase Separation and purification Enzymatic Properties Chemically modified
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 66
Quote: 3
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Abstract


Lipase in the wine industry has a very important significance, Okuma Rhizopus (Rhizopus) is one of the major microbial liquor fermentation process in wine aroma components formed. Luzhou - style and the pros and cons of wine quality, ethyl caproate content of the wine depends primarily on the level of and coordinated with the proportion of other esters. Rhizopus lipase was applied to wine production, but its enzymatic properties of the catalytic mechanism study clarifies, there is a lower enzyme activity, the effect of aroma unstable, strong flavoring targeted, the application of technical conditions is not clear, the use of range not widely practical problems in the wine industry is far from being fully applied. Therefore, in-depth research and clarify Rhizopus lipase enzymatic properties and mechanisms of action of its application in the food industry has great theoretical and practical significance. Rhizopus ZM-10 lipase enzymatic properties and active center amino acid types for the papers to clear targets, the separation and purification of the lipase of Rhizopus ZM-10 conditions, basic enzymatic properties and performance of the kind of activity of essential amino acids main results and conclusions are as follows: the separation and purification of 1. Rhizopus ZM-10 lipase: Rhizopus ZM-10 lipase after ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography, Sephardex G-100 gel filtration layer analysis of lipase, a purification factor of 15.3 times, 22.2% of the activity recovery. Lipase of Rhizopus ZM-10 separation and purification conditions were: the best salting saturation 70%; optimum conditions of DEAE-Sepharose FF anion exchange chromatography: sample volume 5ml, the flow rate of 3mL/min, collecting 3mL per tube, elution volume of 10 column volumes of elution the optimal salt concentration of 0.4mol / L; optimum conditions Sephardex G-100 gel filtration chromatography: sample volume 3mL, flow rate: natural velocity, collecting 1.8 mL per tube, eluted volume: 2 times the column volume. The molecular weight of the lipase was detected by SDS-PAGE gel electrophoresis is approximately 44kDa. Enzymatic properties: 2. Rhizopus ZM-10 lipase purified Rhizopus ZM-10 lipase enzymatic properties: the lipase optimum reaction temperature of 35 ℃, the optimum pH was 8.0. Remains more than 90% of the enzyme at 20 ° C under 1h vitality, has high stability in the pH 7.5,8.0. Na , K , Ca 2 , Mg 2 , Ba 2 and EDTA on its activity was less affected, Zn 2 with significant inhibition. An alcoholic organic solvent has a certain inhibition of the lipase activity, and its stability in the organic solvent of the less polar alkanes. 3. Rhizopus ZM-10 lipase active center of chemical modification: NBS, EDC, DEPC, CH-T, PMSF, DTNB, six kinds of chemical modifying agent chemically modified Rhizopus ZM-10 lipase, substrate protection experiment to study the relationship of the amino acid side chain groups in its molecule with the enzyme active center. Experimental results show that: the acidic amino acids (aspartic acid / glutamic acid) residue, a histidine residue, a serine residue, a tryptophan residue as the essential group Rhizopus ZM-10 lipase activity. Acidic amino acids (aspartic acid / glutamic acid) residue, a histidine residue and a serine residue in the active site of the ZM-10 lipase Rhizopus, and the tryptophan residue in the holder lipase activity from to an important role, but not the active site is located Rhizopus ZM-10 lipase; through EDC DEPC modification of kinetic experiments show that molecules active center of the lipase of Rhizopus ZM-10 include at least an aspartic acid (or Valley leucine) residue and a histidine residue.

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CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )
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