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 improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have too low a content of surface lysine groups to permit effective crosslinking with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The co-aggregation 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). First, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during the precipitation and crosslinking steps nearly doubled the recovered activity. Similar recovered activity (around 40%) was achieved when 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). Moreover, 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 filtration or centrifugation recovery methods usually used.
Highlights
Amyloglucosidase (1,4-α-D-glucan hydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of glucose from the non-reducing ends of glucose polysaccharides
Because the biochemical and structural properties differ from one enzyme to another, a screening of precipitants should be performed for the preparation of Cross-Linked Enzyme Aggregates (CLEAs) of a particular enzyme [20,45]
For the CLEAs prepared in the presence of PEI, the immobilization yields were higher than 95% for all conditions, but the recovered activity were higher when CLEAs were prepared under gently stirring in 3D Platform Shaker
Summary
Amyloglucosidase (1,4-α-D-glucan hydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of glucose from the non-reducing ends of glucose polysaccharides. Catalysts 2018, 8, x FOR PEER REVIEW but in a slower manner. Like the ones produced by Aspergillus niger glycosidic bonds), but in[1,2]. A slower manner [1,2]. Like the ones produced (AMG), may present more than one form, with different molecular weights [3,4,5]. Aspergillus niger by Aspergillus niger (AMG), may present more than one form, with different molecular weights [3,4,5]. G1 isoform, which to acorresponds protein having catalytichaving domain. G1corresponds isoform, which to aa protein a 1), and a starch-binding domain (structure on the right in 1)
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