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Molecules 2018, 23(12), 3188; https://doi.org/10.3390/molecules23123188

Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

1
Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain
2
Department 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, Bejaia 06000, Algeria
*
Author to whom correspondence should be addressed.
Received: 21 November 2018 / Revised: 29 November 2018 / Accepted: 30 November 2018 / Published: 3 December 2018
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Abstract

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 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, 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 inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67 °C, 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 45 °C and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity.
Keywords: enzyme immobilization; enzyme stabilization; solid phase chemical modification; enzyme amination; glutaraldehyde; crosslinking enzyme immobilization; enzyme stabilization; solid phase chemical modification; enzyme amination; glutaraldehyde; crosslinking
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Hussain, F.; Arana-Peña, S.; Morellon-Sterling, R.; Barbosa, O.; Ait Braham, S.; Kamal, S.; Fernandez-Lafuente, R. Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde. Molecules 2018, 23, 3188.

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