Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk.
The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
César
C
Mateo
Rubens
R
Monti
Benevides C C
BC
Pessela
Manuel
M
Fuentes
Rodrigo
R
Torres
José Manuel
JM
Guisán
Roberto
R
Fernández-Lafuente
D000818:Animals; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D043322:Lactase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D008892:Milk / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D043322:Lactase; D005947:Glucose; D007785:Lactose