Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent

Abstract

Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration of the crosslinker) were evaluated on the production of CLEAs of CGTase. Among the seven studied precipitants, acetone with a 75% (v/v) concentration produced the aggregates of CGTase with higher activity, which retained 97% of the initial activity. Concerning the cross-linker (glutaraldehyde, starch–aldehyde, and pectin–aldehyde), starch–aldehyde produced the most active CLEAs. The use of bovine serum albumin as co-feeder decreased the expressed activity. Addition of polyethylenimine at the end of cross-linking step prevented the leakage of the enzyme and the subsequent Schiff’s bases reduction with sodium borohydride permitted to maintain 24% of the initial activity even with the large dextrin as substrate. The optimal conditions for the immobilization process required were defined as 75% (v/v) acetone as precipitation reagent for 1 h at 20 °C, 20 mM starch–aldehyde as crosslinking reagent for 2 h at 20 °C, treatment with 1 mg/mL of polyethylenimine for 5 min, reduction with 1 mg/mL of sodium borohydride. The CLEAs of CGTase were active catalyst (similarly to the free enzyme) in the production of cyclodextrins at 50 °C and pH 6.0 for 6 h reaction, maintaining intact their structures. Besides this, after five cycles of 3 h the total cyclodextrin yield was 80% of the initial value (first batch, with around 45% CD yield).

Parameters necessary to define an immobilized enzyme preparation

“…This may be determined by measuring the activity of the suspension and supernatants in each washing step. The solution may be to add media rich in primary amino groups, proteins or polymers, amination of the enzyme or alternatively use other cross-linkers (159,160,161,162,163,164,165,166,167,168).…”

Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

“…The presence of a non-catalytic carrier within the immobilized enzyme matrix further reduces the volumetric catalyst [20][21][22]. Eliminating the need to incorporate any solid support as a carrier maximizes the catalytic performance and reduces the cost of preparing the CLEA since the cross-linkers are relatively small and inexpensive [23,24].…”

Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermophilus

“…However, the xylanase activity was only preserved in the CLEAs prepared at pH 8.5. The xylanase activity lost in the CLEAs prepared with glutaraldehyde at pH 6.5 can be attributed to the diffusional problem of xylan with the supramolecular structure [ 27 ]. In agreement with this, the diffusional problems could minimize if the spatial structure of CLEAs is enlarged.…”