Preparation of immobilized/stabilized biocatalysts of β‐glucosidases from different sources: Importance of the support active groups and the immobilization protocol

Abstract

β‐Glucosidases from two different commercial preparations, Pectinex Ultra SP‐L and Celluclast® 1.5L, were immobilized on divinylsulfone (DVS) supports at pH 5.0, 7.0, 9.0, and 10. In addition, the biocatalysts were also immobilized in agarose beads activated by glyoxyl, and epoxide as reagent groups. The best immobilization results were observed using higher pH values on DVS‐agarose, and for Celluclast® 1.5L, good results were also obtained using the glyoxil‐agarose immobilization. The biocatalyst obtained using Pectinex Ultra SP‐L showed the highest thermal stability, at 65°C, and an operational stability of 67% of activity after 10 reuses cycles when immobilized on DVS‐agarose immobilized at pH 10 and blocked with ethylenediamine. The β‐glucosidase from Celluclast® 1.5L produced best results when immobilized on DVS‐agarose immobilized at pH 9 and blocked with glycine, reaching 7.76‐fold higher thermal stability compared to its free form and maintaining 76% of its activity after 10 successive cycles. The new biocatalysts obtained by these protocols showed reduction of glucose inhibition of enzymes, demonstrating the influence of immobilization protocols, pH, and blocking agent.

High-Purity Preparation of Enzyme Transformed Trans-Crocetin Reclaimed from Gardenia Fruit Waste

“…Celluclast ® 1.5 L has been shown a good tolerance to glucose at pH 3.0 and a low Km value [ 29 ]. These characteristics were supposed to be capable for application in the deglucosylation of crocins in the study.…”

“…Considering the screening of commercial enzyme preparations containing β-glucosidase activity, the Celluclast ® 1.5 L was chosen to apply the hydrolysis study of crcins from nine commercial products as described previously [ 29 ]. In brief, the crocins were hydrolyzed using Celluclast ® 1.5 L at 2:1 ratio ( w / w ) as per the instructions by the manufacture.…”

Immobilization-Stabilization of β-Glucosidase for Implementation of Intensified Hydrolysis of Cellobiose in Continuous Flow Reactors

“…These results agree with those reported in the literature when the β-glucosidase was immobilized in a similar support (mainly agarose) or similar immobilization chemistry was employed: high immobilization yields and recovered activities were obtained for the reversible immobilization [45][46][47], while lower yields for irreversible immobilization were attained [45,48]. The choice between PEI-activated and glyoxyl-activated requires further research according to the performance in terms of kinetic, productivity, and operational stability, which was studied, and it is described as follows [17,18].…”

“…One strategy to facilitate the effective β-glucosidase implementation is the design and implementation of the enzyme in the format of enzyme-immobilized preparations [17,18]. Enzyme immobilization is an enabling technology that facilitates enzyme handling, permits its cyclic reuse and continuous use, and, if properly designed, might contribute to increasing the operational stability of the enzyme [19,20].…”

“…Active immobilization by adsorption (anion exchange based ionic adsorption) or covalent binding (via aldehyde chemistry) to porous material supports have been shown as a promising strategy to obtain active and stable catalysts of different βglucosidases. In these previous works, the focus of the immobilization design was obtaining high specific activity (similar to the free enzyme) and enhancing the stability under resting conditions, which represents a promising point for implementation [17,18,[24][25][26][27][28][29][30][31][32].…”