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

Abstract

Cellulose saccharification to glucose is an operation of paramount importance in the bioenergy sector and the chemical and food industries, while glucose is a critical platform chemical in the integrated biorefinery. Among the cellulose degrading enzymes, β-glucosidases are responsible for cellobiose hydrolysis, the final step in cellulose saccharification, which is usually the critical bottleneck for the whole cellulose saccharification process. The design of very active and stable β-glucosidase-based biocatalysts is a key strategy to implement an efficient saccharification process. Enzyme immobilization and reaction engineering are two fundamental tools for its understanding and implementation. Here, we have designed an immobilized-stabilized solid-supported β−glucosidase based on the glyoxyl immobilization chemistry applied in porous solid particles. The biocatalyst was stable at operational temperature and highly active, which allowed us to implement 25 °C as working temperature with a catalyst productivity of 109 mmol/min/gsupport. Cellobiose degradation was implemented in discontinuous stirred tank reactors, following which a simplified kinetic model was applied to assess the process limitations due to substrate and product inhibition. Finally, the reactive process was driven in a continuous flow fixed-bed reactor, achieving reaction intensification under mild operation conditions, reaching full cellobiose conversion of 34 g/L in a reaction time span of 20 min.

Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis

“…Thus, immobilization reduces, if not eliminates, this inhibition. This inhibitory effect, however, was also observed in a recent paper by our group when working with cross-linked agarose beads activated with polyethylenemine (PEI), or glyoxyl groups (Gly) [14], suggesting that support and immobilization chemistry play a role in the inhibitory role of cellobiose. The kinetic models proposed, and fitted to all data, are Michaelis-Menten models with competitive inhibition by glucose, a type of inhibition very common to hydrolases acting on poly-, oligo-and disaccharides.…”

“…Due to its industrial potential, there are many recent reports on fungal β-glucosidase immobilization on different supports by several immobilization methods such as the following: β-glucosidase of Aspergillus versicolor immobilized on magnetic MnO 2 nanomaterials [8], β-glucosidase from Aspergillus niger immobilized on diverse amino agarose beads and cross-linked with glutaraldehyde [9], β-glucosidase from Aspergillus awamori using commercial gelatin as support and glutaraldehyde as crosslinker [10], β-glucosidase of Aspergillus niger on amino-based silica via biotin-streptavidin affinity [11], β-glucosidase of Aspergillus niger immobilized on Eupergit ® C (an epoxy-activated support) [12], βglucosidase of Aspergillus japonicus immobilized on anionic exchanger supports (MANAEagarose and DEAE-cellulose) [13], and β-glucosidase of Aspergillus fumigatus immobilized on cross-linked agarose beads and polymethacrylate Lifetech™ECR8209F particles (Purolite) activated with amino groups (monoaminoethyl-N-aminoethyl: MANAE), polyethylenemine (PEI), or glyoxyl groups (Gly) [14], to name a few. Among all types of immobilization methods, covalent immobilization is the most used in industry, and different activated supports for that purpose have been developed, based on Eupergit ® , Sepabeads™ and ReliZyme™.…”

Development of a Multi-Enzymatic Biocatalytic System through Immobilization on High Quality Few-Layer bio-Graphene

“…We also tested the tri-enzymatic nanobiocatalyst for the cascade where cellobiose hydrolysis is the initial step; the results for different reaction temperatures are shown in Figure 12 b. The highest catalytic activity was observed at 50 °C, which is known to be the optimal operating temperature for β -glucosidases towards cellobiose hydrolysis [ 48 ]. It is interesting to note that contrary to p NPG hydrolysis, in this cascade reaction, the tri-enzymatic nanobiocatalytic system is effective at 50 °C.…”

What’s new in flow biocatalysis? A snapshot of 2020–2022

“…PBRs are easily scalable and applied for large-scale continuous biotransformations in diverse industry sectors, from the well-known resolution of acyl-DLamino acids employing the immobilized Aspergillus oryzae aminoacylase (Sato and Tosa, 2010), to the most recent production of biodiesel (Erdem and Woodley, 2022;Miotti et al, 2022) or fructose syrup (Neifar et al, 2020). Further recent applications include the continuous removal of urea in high polyphenol wines with an immobilized Lactobacillus fermentum acid urease to reduce ethyl carbamate formation (Fidaleo and Tavilli, 2021), and the hydrolysis of cellobiose with immobilized βglucosidases (Alvarez-Gonzalez et al, 2022). Cellobiose, a β-1,4 glucose-based disaccharide, is used as a model-molecule in many studies aimed at finding solutions to the depolymerization of cellulose from renewable lignocellulosic biomass.…”

“…Bolivar and coworkers recently reported on a sustainable cellobiose hydrolysis. By designing a glucosidase-based reactor using glyoxyl-agarose for immobilization, the full conversion (34 g L -1 ) was achieved in 20 min residence time (Alvarez-Gonzalez et al, 2022). An engineered variant of the hydrolytic enzyme β-N-acetyl-hexosaminidase from Bifidobacterium bifidum was employed to produce the precious oligosaccharide lacto-N-triose II, a component of human milk oligosaccharides used as a synthon for nutritional supplements, providing a further example of the synergistic use of protein and reaction engineering for synthetic purposes (Ruzic et al, 2020).…”