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

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.

Recent Strategies and Applications for l-Asparaginase Confinement

“…The stability of the chemical bond is defined by the enzyme binding direction, achieving maximum activity levels when the active centre amino acids are not involved in the support bonding [66,95]. The support linkage is established either via reactive functional groups already present in the support or through support modification to produce activated groups [66,95,114,115]. way, both enzymes after confinement showed less affinity for the substrate.…”

“…The stability of the chemical bond is defined by the enzyme binding direction, achieving maximum activity levels when the active centre amino acids are not involved in the support bonding [66,95]. The support linkage is established either via reactive functional groups already present in the support or through support modification to produce activated groups [66,95,114,115]. Silica has been broadly used as an inert and stable support for enzyme confinement due to its tuneable physicochemical characteristics, such as tailorable pore diameters, which may vary from microporous (<2 nm), mesoporous (2-50 nm) or macroporous (>50 nm) silicas, depending on the confined enzyme dimension (3 to 6 nm) [116][117][118].…”

Editorial for Special Issue: Enzyme Immobilization and Its Applications

“…In another paper, an indirect yeast surface display method was described by anchoring Im7 proteins on the surface of Pichia pastoris, using this system to immobilize fluorescence proteins (sfGFP and mCherry) and enzymes (human arginase I) where a CL7 fusion tag has been introduced [36]. Chemical modification (amination of the enzyme surface plus glutaraldehyde treatment) of Alcalase previously immobilized on glyoxyl agarose beads permitted significant improvement of enzyme stability, enabling the use of the enzyme at higher temperatures than the nonmodified enzyme or the free enzyme [37]. Superfolder green (sGFP) and red (RFP) fluorescent proteins have been fused with His-tags to immobilize them on graphene 3D hydrogels, with Cys-tags to immobilize them on porous matrices activated with either epoxy or disulfide groups or with Lys-tags to immobilize them on nanoparticles functionalized with carboxylic groups [38], to spatially tune the protein distribution.…”

Development of protease nanobiocatalysts and their application in hydrolysis of sunflower meal protein isolate

“…In this research, we report the development of reliable and efficient Alcalase ® 2.4 L and Flavourzyme ® nanobiocatalyst systems. Alcalase ® 2.4 L is a commercial crude preparation produced by a selected strain of Bacillus licheniformis and its main component is a serine protease (subtilisin Carlsberg) with endopeptidase activity (Hussain et al ., 2018). Flavourzyme ® is a nonspecific protease preparation obtained by fermentation of Aspergillus oryzae strain which consists of 8 enzymes with both endoprotease and exopeptidase activities (Merz et al ., 2015).…”