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Preparation of magnetic cross-linked amyloglucosidase aggregates: solving some activity problems

Autores:Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
Murilo Amaral-Fonseca 1, Willian Kopp 2, Raquel de Lima Camargo Giordano 1, Roberto Fernández-Lafuente 3,* and Paulo Waldir Tardioli 1,*
1   Postgraduate Program in Chemical Engineering, Department of Chemical Engineering, Federal University of São Carlos (PPGEQ/UFSCar), Rod. Washington Luiz, km 235, 13565-905, São Carlos, SP, Brazil; muriloaf88@hotmail.com (M. Amaral-Fonseca); raquel@ufscar.br (R. L. C. Giordano); pwtardioli@ufscar.br (P. W. Tardioli).
2   Kopp Technologies (KTech), Rua Alfredo Lopes, 1717, Jardim Macarengo, 13560-460, São Carlos, SP, Brazil; solutions@kopptechnologies.com (W. Kopp).
3   Departamento de Biocatális, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain; rfl@icp.csic.es (R. Fernandez-Lafuente).

Abstract: The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and with improved stabilities. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have a too low content of surface lysine groups to permit effective crosslink with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The coaggregation with magnetic particles makes their handling easier. In this work, CLEAs of a commercial amyloglucosidase (AMG) produced by Aspergillus niger were prepared by co-aggregation in the presence of polyethyleneimine (PEI) or starch with aminated magnetic nanoparticles (MNPs) or bovine serum albumin (BSA). Firstly, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during precipitation and crosslinking steps increased the recovered activity around twofold. Similar recovered activity (around 40%) was achieved changing starch by PEI. Moreover, under the same conditions, AMG co-aggregated with BSA was also synthesized, yielding CLEAs with very similar recovered activity. Both CLEAs (co-aggregated with MNPs or BSA) were four times more stable than the soluble enzyme. These CLEAs were evaluated in the hydrolysis of starch at typical industrial conditions, achieving more than 95% starch-to-glucose conversion measured as Dextrose Equivalent (DE). Besides, both CLEAS could be reused for five cycles maintaining a DE of around 90%. Although both CLEAs had good properties, magnetic CLEAs could be more attractive for industrial purposes because of their easy separation by an external magnetic field, avoiding the formation of clusters during the usually used filtration or centrifugation recovery methods.
Palabras clave:cross-linked enzyme aggregate; amyloglucosidase; magnetic nanoparticles; bovine serum albumin; polyethyleneimine; starch hydrolysis
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