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.
Highlights
Proteases are enzymes with many uses in biotechnology, from hydrolysis of proteins to reduction of allergenicity or production of bioactive peptides, to fine chemistry being the detergent industry the main consumer of proteases [1,2,3,4,5,6,7,8].Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis
Immobilization of Alcalase in Glyoxyl Agarose most enzyme activity was immobilized and expressed activity was over 50%
Alcalase immobilized on glyoxyl agarose offers some activity versus casein and a significant stabilization, lower using agarose 4BCL than the previously described values using agarose 6BCL, as may be expected from the different geometry of the support [64]
Summary
Proteases are enzymes with many uses in biotechnology, from hydrolysis of proteins to reduction of allergenicity or production of bioactive peptides, to fine chemistry (production of dipeptides, resolution of racemic mixtures, etc.) being the detergent industry the main consumer of proteases [1,2,3,4,5,6,7,8].Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis. One of the most pursued objectives of immobilization is the increase of enzyme stability, via multipoint or multisubunit covalent attachment, enzyme activity (mainly under harsh conditions), specificity or selectivity, resistance to inhibitors and chemicals or enzyme purity may be improved via a proper immobilization [21,22,23,24,25,26,27,28,29] It has been recently described how diffusion limitations generated via immobilization on porous supports may be useful in tailoring protease selectivity [30,31]. This makes compulsory to have a proper orientation of the active centre of the enzyme, which should be oriented towards the medium, otherwise the enzyme may become inactive just by steric reasons even having the structure of the active centre unaltered [32]
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