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Immobilization of Lactase from Kluyveromyces lactis Greatly Reduces the Inhibition Promoted by Glucose. Full Hydrolysis of Lactose in Milk
Author(s) -
Mateo César,
Monti Rubens,
Pessela Benevides C. C.,
Fuentes Manuel,
Torres Rodrigo,
Manuel Guisán José,
FernándezLafuente Roberto
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049957m
Subject(s) - chemistry , glutaraldehyde , non competitive inhibition , lactose , hydrolysis , kluyveromyces lactis , enzyme , substrate (aquarium) , enzyme assay , enzyme kinetics , chromatography , immobilized enzyme , biochemistry , active site , yeast , saccharomyces cerevisiae , oceanography , geology
The kinetic constants ( K m , V max , and inhibition constants for the different products) of soluble and different immobilized preparations of β‐galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the K m 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 K m 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 ( K i ≈ 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.