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RevistaMolecules
Año2018
Volumen23
Páginas3188
Internacional

Further stabilization of Alcalase immobilized on glyoxyl supports: amination plus modification with glutaraldehyde.

Autores:Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
Fouzia Hussain1, 2, Sara Arana-Peña1, Roberto Morellon-Sterling1, Oveimar Barbosa3, Sabrina Ait Braham1, 4, Shagufta Kamal2, Roberto Fernandez-Lafuente1,*
1   1Departamento de Biocatálisis. ICP-CSIC, Campus UAM-CSIC Madrid, Spain
2   2Department of Biochemistry, Government College University, Faisalabad 38000, Pakistan.
3   Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué 546,  Colombia.
4   Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Algeria

 
Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc‐L‐alanine 4‐nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50ºC, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60ºC, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivations at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67ºC, retaining around 50% of the activity after 5 hydrolytic cycles when the just immobilized preparation was almost inactive after 3 cycles. The modified enzyme can be reused in hydrolysis of casein at 45ºC and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity.
 
Palabras clave:Enzyme stabilization, Enzyme immobilization, glutaraldehyde, crosslinking, enzyme amination, solid phase chemical modification
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