The enzymatic conversion of biomass-derived compounds represents a key step in the biorefinery flowsheet, allowing low-temperature high-efficiency reactions. ?-Glucosidases are able to hydrolyze cellobiose into glucose. Wrinkled silica nanoparticles (WSNs) were demonstrated to be a good support for the immobilization of ?-glucosidases, showing better performance than free enzymes in batch reaction; on the other hand, immobilized enzyme microreactors (IEMs) are receiving significant attention, because small quantities of reagents can be used, and favorable heat and mass transfer can be achieved with respect to conventional batch systems. In this work, we prepared, characterized, and tested structured enzymatic reactor compounds by a honeycomb monolith, a WSN washcoat, and ?-glucosidases as the active phase. Powder and structured materials were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), N physisorption, thermogravimetric analysis (TGA), and Fourier-transform infrared spectroscopy (FT-IR). Structured catalysts were tested under both batch and continuous flow reaction conditions and compared to powder catalysts (batch reaction). The WSN washcoat was attached well onto the monolith walls, as suggested by the negligible weight loss after ultrasound treatment; the WSNs preserved their shape, porosity, and individual nature when deposited onto the monolith walls. The immobilized enzyme microreactors proved to be very efficient in hydrolysis of cellobiose to glucose, showing a complete conversion under continuous flow reaction at a batch-equivalent contact time equal to 120 min vs. 24 h obtained in the batch experiments. The apparent K value showed a 20-fold decrease with respect to the batch process, due to the absence of external diffusive transport limitations.

The enzymatic hydrolysis of lignocellulosic biomass-derived compounds represents a valid strategy to reduce the dependence on fossil fuels, with geopolitical and environmental benefits. In particular, ?-glucosidase (BG) enzyme is the bottleneck in the degradation of cellulose because it catalyzes the hydrolysis of cellobiose, a known inhibitor of the other cellulolytic enzymes. However, free enzymes are unstable, expensive and difficult to recover. For this reason, the immobilization of BG on a suitable support is crucial to improve its catalytic performance. In this paper, computational fluid dynamics (CFD) simulations were performed to test the hydrolysis reaction in a monolith channel coated by BG adsorbed on a wrinkled silica nanoparticles (WSNs) washcoat. We initially defined the physical properties of the mixture, the parameters related to kinetics and mass transfers and the initial and boundary conditions thanks to our preliminary experimental tests. Numerical simulation results have shown great similarity with the experimental ones, demonstrating the validity of this model. Following this, it was possible to explore in real time the behavior of the system, varying other specified parameters (i.e., the mixture inlet velocity or the enzymatic load on the reactor surface) without carrying out other experimental analyses.

An immobilization protocol of a model enzyme into silica nanoparticles was applied. This protocol exploited the use of the bifunctional molecule triethoxysilylpropylisocyanate (TEPI) for covalent binding through a linker of suitable length. The enzyme ?-glucosidase (BG) was anchored onto wrinkled silica nanoparticles (WSNs). BG represents a bottleneck in the conversion of lignocellulosic biomass into biofuels through cellulose hydrolysis and fermentation. The key aspect of the procedure was the use of an organic solvent (anhydrous acetone) in which the enzyme was not soluble. This aimed to restrict its conformational changes and thus preserve its native structure. This approach led to a biocatalyst with improved thermal stability, characterized by high immobilization efficiency and yield. It was found that the apparent KM value was about half of that of the free enzyme. The Vmax was about the same than that of the free enzyme. The biocatalyst showed a high operational stability, losing only 30% of its activity after seven reuses.

?-Glucosidase (BG) was immobilized by adsorption on wrinkled silica nanoparticles (WSNs) and on tannic acid-templated mesoporous silica nanoparticles (TA-MSNPs). The effect induced by a different morphology of the pores of the sorbent on the catalytic performance of ?-glucosidase was investigated. A complete textural and morphological characterization of the two samples was performed by Brunauer-Emmett-Teller (BET) method, Fourier Transform Infrared (FT-IR) and transmission electron microscopy (TEM). The results demonstrated that the catalytic performance of the immobilized enzyme depends on the pores size of sorbent but a key factor is the pores morphology. In fact, the BG immobilized on WSNs and TA-MSNPs (BG/WSNs and BG/TA-MSNPs) shows in both cases good catalytic performances in cellobiose hydrolysis, but the catalyst with the best performance is BG/WSNs, in which the support exhibits a central-radial pore structure and a hierarchical trimodal micro-mesoporous pore size. This peculiar morphology allows the enzyme to settle in a place where the interactions with the walls are maximized, increasing its conformational rigidity. Furthermore, the enzyme is prevalently collocated in the interior of pore so that the pores are not completely capped.

Forty-one strains of lactic acid bacteria (LAB) isolated from Cornetto di Matera sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process. Urease, peptidase, phytase, phosphatase and ?-glucosidase activities were measured spectrophotometrically using synthetic substrates. Proteolytic activities were examined in model doughs, using neutral and acidified sterile doughs as controls. All strains had low urease, glutamyl aminopeptidase and iminopeptidase activities, whereas differences within species were observed for the other enzymatic activities. Leuconostoc mesenteroides and Lactobacillus curvatus strains generally showed high aminopeptidase, X-prolyl dipeptidyl aminopeptidase, ?-glucosidase and phytase activities, while the enzymatic activities of Lactobacillus plantarum, Lactobacillus pentosus and Weissella cibaria varied between strains. In order to classify the strains on the basis of similar enzymatic profiles, a hierarchical cluster analysis was carried out. Several strains of L. plantarum, L. curvatus and Leuc. mesenteroides showed an interesting combination of proteolytic, peptidase, ?-glucosidase and phytase activities, suggesting their possible usefulness as a mixed starter culture in bread-making processes. © 2006 Elsevier B.V. All rights reserved.

The ?-glucosidase from olive fruit is of particular interest compared to the ones from other sources because it has shown to have high specifity to convert the oleuropein into dialdehydes, which have antibacterial activity and are of high interest for their application in the food and pharmaceutical fields. The enzyme is not yet commercially available and advanced clean and safe technologies for its purification able to maintain the functional stability are foreseen. The purification of this protein from fruit extracts has been already tempted by electrophoresis but either enzyme deactivation or high background with unclear profiles occurred. In this work, fruit extracts obtained from the ripening stage that showed the highest enzyme activity have been processed by diafiltration and ultrafiltration. Asymmetric membranes made of polyamide or polysulphone having 50 and 30 kDa molecular weight cut-off, respectively, were tested for the diafiltration process. Ultrafiltration membranes made of polyethersulfone with 4 kDa molecular weight cut-off were used to concentrate the diafiltered permeate solutions. The efficiency of the separation processes was evaluated byenzyme activity tests using the hydrolysis of p-D-nitrophenyl-?-D-glucopyranoside (pNPGlc) as reaction model. Qualitative and quantitative electrophoresis were applied to analyze the composition of protein solution before and after the membrane separation; in addition dot blot and western blot analyses were applied to verify the presence of ?-glucosidase in the processed fractions. The overall results showed that the ?-glucosidase functional stability was preserved during the membrane operations and the removal of 20 kDa proteins allowed to increase the specific activity of the enzyme of about 52% compared to the one present in the initial fruit extract.