[Immobilization of lipase labeled with fluorescent probe and its stability].
The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.
Xu
Jiayin
J
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Zhang
Chi
C
Song
Xijin
X
Wang
Jie
J
D003911:Dextrans / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D058767:Protein Unfolding / Q000187:drug effects
D003911:Dextrans; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; C015219:fluorescein isothiocyanate dextran; D011092:Polyethylene Glycols; D008049:Lipase; D016650:Fluorescein-5-isothiocyanate