Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme.
Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.
Montero
S
S
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad del País Vasco, Bilbao, Spain.
Blanco
A
A
Virto
M D
MD
Landeta
L C
LC
Agud
I
I
Solozabal
R
R
Lascaray
J M
JM
de Renobales
M
M
Llama
M J
MJ
Serra
J L
JL
D001709:Biotechnology; D002175:Candida / Q000201:enzymology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D009994:Osmolar Concentration; D012995:Solubility; D013696:Temperature
D004800:Enzymes, Immobilized; D008049:Lipase