Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

Abstract

Establishing a suitable immobilization strategy to improve the accessibility of immobilized cellulase for insoluble cellulose and subsequently recover the enzyme from the remaining substrate is crucial to promote the industrialization of biofuels. Here, using iminodiacetic acid (IDA) and Ni2+ ions, a novel metal-chelated copolymer of methacrylic acid and methyl methacrylate was prepared for the immobilization of His-tagged processive endoglucanase EG5C-1 via affinity interaction between polyhistidine tag and Ni2+. The optimum loading capacity of the functionalized polymer (Eud-IDA-Ni2+) for EG5C-1 was about 280 mg/g, where more than 80% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal and pH stability and better reusability than the free one, and after five cycles of usage, the hydrolysis productivity remained above 70% of the initial value. The Eud-IDA-Ni2+/EG5C-1 biocomposite displayed reversibly soluble–insoluble characteristics with pH change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the pH after the reaction. Thus, the yield obtained from the hydrolysis of an insoluble phosphoric acid-swollen cellulose substrate was similar for the free and immobilized form of EG5C-1. Our combined results suggested that a metal-ion-chelated, pH-responsive polymer had the potential for His-tagged cellulase immobilization to improve both the operational efficiency and economic benefit of the cellulosic biorefinery industry.

Immobilization and Characterization of a Processive Endoglucanase EG5C-1 from Bacillus subtilis on Melamine–Glutaraldehyde Dendrimer-Functionalized Magnetic Nanoparticles

“…The construction, expression, and purification of C-terminally His-tagged EG5C-1 were carried out according to our previous methods [ 28 ]. The purified enzyme was assayed by SDS-PAGE and freeze-dried for storage.…”

Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses

“…Therefore, RfGH5_4 endoglucanase not only reduces the need of cellobiohydrolase to generate cellobiose during synergistic saccharification but it will also boost the saccharification process even if an additional cellobiohydrolase is used. RfGH5_4 could also be immobilized using metal ion assisted recyclable pH-responsive polymer like Eudragit S100 to hydrolyse lignocellulose in bioethanol industry [55]. Moreover, RfGH5_4 is sufficiently capable of hydrolysing hemicellulosic polysaccharides as described in Section 3.5.2 which further decreases the requirement of additional hemicellulases.…”

Cellulase Immobilization on Nanostructured Supports for Biomass Waste Processing

“…It is noteworthy that one of the problems of catalyzing the hydrolysis of cellulosic biomass with immobilized cellulase is interactions of two solids. To counteract this shortcoming, an interesting strategy for the development of the support was utilized by modification of a copolymer of methacrylic acid and methyl methacrylate which is pH-responsive and allows a phase transition between soluble and insoluble forms [ 141 ]. A functionalization of this polymer with iminodiacetic acid (IDA) and Ni 2+ ions created immobilization points for His -tagged endoglucanase EG5C-1 (cellulase) due to its affinity interaction with Ni 2+ ( Figure 10 ).…”

“… Schematic representation of the recyclable Eud-IDA-Ni 2+ /EG5C-1 biocomposite which is soluble at pH above 5.5 and insoluble for separation at pH below 4.5, can effectively hydrolyze cellulose. Reproduced with permission from [ 141 ], American Chemical Society, 2021. …”