Characterization of cross-linked enzyme aggregates (CLEAs) of the fusion protein FUS-PepN_PepX and their application for milk protein hydrolysis

Preparation and Characterization of Sugar-Assisted Cross-Linked Enzyme Aggregates (CLEAs) of Recombinant Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE)

“…The conventional preparation of CLEAs usually includes the aggregation and precipitation of target enzyme proteins from aqueous buffer (step 1) followed by cross-linking with bifunctional agents (step 2). , This kind of carrier-free method has several advantages over conventional carrier-bound immobilization, such as high activity volumes and low production cost due to the exclusion of inert carriers. , However, both steps may cause an alteration of enzyme configuration, thus leading to a significant reduction in expressed activity of CLEAs. ,, The using of water-miscible organic solvents or a high amount ammonium sulfate in step 1 may induce the denaturation of enzyme proteins due to the stripping of the crucial water layer from the enzyme surface. , As to step 2, the formation of Schiff bases between aldehyde groups of glutaraldehyde and amino groups of enzyme proteins could increase the rigidity (mechanical resistance) of CLEAs and thus enhance its thermal stability, but the enzyme might undergo a change in conformation if the cross-linking affects the active sites. , …”

“…More importantly, CLEAs maintained almost 100% activity when incubated at ≤65 °C for 12 h, whereas the value for free WT- Cs CE was only 78% at 65 °C. This is a great advantage because CLEAs with higher catalytic activity (Figure b) and significantly enhanced thermal stability at lower temperatures (<75 °C) (Figure d) as well as excellent reusability at 60 °C (Figure ) will obviously favor the industrial biosynthesis of lactulose, since lower temperatures (<75 °C) are more applicable for industrial applications in terms of energy savings and product safety. ,, However, it should be pointed out that at higher temperatures (≥75 °C) CLEAs were even less stable than free WT- Cs CE (Figure d). The reason for this is still unclear.…”

“…13,17 However, both steps may cause an alteration of enzyme configuration, thus leading to a significant reduction in expressed activity of CLEAs. 12,16,21 The using of water-miscible organic solvents or a high amount ammonium sulfate in step 1 may induce the denaturation of enzyme proteins due to the stripping of the crucial water layer from the enzyme surface. 15,20 As to step 2, the formation of Schiff bases between aldehyde groups of glutaraldehyde and amino groups of enzyme proteins could increase the rigidity (mechanical resistance) of CLEAs and thus enhance its thermal stability, but the enzyme might undergo a change in conformation if the cross-linking affects the active sites.…”

“…The use of immobilized biocatalysts confers many advantages (enhanced stability, robust reusability, simplified downstream processing, etc.) over purified enzymes in many bioconversion processes. , Carrier-free immobilized enzymes, particularly cross-linked enzyme aggregates (CLEAs), have attracted extensive attention due to their high enzyme activity, good specificity, low production cost, and easy handling when compared with conventionally immobilized enzymes (carrier bound and encapsulation/entrapment). , Generally, the preparation of CLEAs often involves the precipitation/aggregation of soluble enzymes (not necessarily in a pure form) by inorganic salts, organic solvents, or nonionic polymers, followed by cross-linking of the resulting enzyme aggregates, Figure . ,, However, a potential drawback of such a method is that alteration of enzyme configuration might occur during the aggregation and cross-linking processes, which may eventually lead to reduced enzyme activity. , This drawback can be overcome by aggregating and cross-linking the soluble enzymes in the presence of certain protective additives such as bovine serum albumin (BSA) − or sugars. , In recent years, the CLEAs technique has been successfully applied to a broad range of enzymes, including acylase, α-amylase, laccase, β-galactosidase, etc. ,,, However, there is only one report on synthesizing lactulose by CLEAs of β-galactosidase in repeated-batch operation …”

“…17,20 In recent years, the CLEAs technique has been successfully applied to a broad range of enzymes, including acylase, α-amylase, laccase, β-galactosidase, etc. 14,15,21,22 However, there is only one report on synthesizing lactulose by CLEAs of β-galactosidase in repeated-batch operation. 16 To the best of our knowledge, there is no report in the literature dealing with the use of CLEAs of WT-CsCE for lactulose biosynthesis.…”