Carrier-free immobilization of lipase from Candida rugosa with polyethyleneimines by carboxyl-activated cross-linking.
Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs' size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 °C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.
Departamento de Biotecnología, Universidad Autónoma Metropolitana Iztapalapa , Avenida San Rafael Atlixco #186, Col. Vicentina 09340, Distrito Federal México.
Susana
S
Velasco-Lozano
Fernando
F
López-Gallego
Rafael
R
Vázquez-Duhalt
Juan C
JC
Mateos-Díaz
José M
JM
Guisán
Ernesto
E
Favela-Torres
D000818:Animals; D002175:Candida / Q000201:enzymology*; D002234:Carbodiimides / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010316:Particle Size; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013696:Temperature
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D012710:Serum Albumin, Bovine; D011094:Polyethyleneimine; D008049:Lipase