Lipases are ubiquitous enzymes whose physiological role is the hydrolysis of triacylglycerol into fatty acids. They are the most studied and industrially interesting enzymes, thanks to their versatility to promote a plethora of reactions on a wide range of substrates. In fact, depending on the reaction conditions, they can also catalyze synthesis reactions, such as esterification, acidolysis and transesterification. The latter is particularly important for biodiesel production. Biodiesel can be produced from animal fats or vegetable oils and is considered as a biodegradable, non-toxic and renewable energy source. The use of lipases as industrial catalysts is subordinated to their immobilization on insoluble supports, to allow multiple uses and use in continuous processes, but also to stabilize the enzyme, intrinsically prone to denaturation with consequent loss of activity. Among the materials that can be used for lipase immobilization, mesoporous silica nanoparticles represent a good choice due to the combination of thermal and mechanical stability with controlled textural characteristics. Moreover, the presence of abundant surface hydroxyl groups allows for easy chemical surface functionalization. This latter aspect has the main importance since lipases have a high affinity with hydrophobic supports. The objective of this work is to provide an overview of the recent progress of lipase immobilization in mesoporous silica nanoparticles with a focus on biodiesel production.

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 β-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 K_M value was about half of that of the free enzyme. The V_max 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.

Mesostructured silica nanoparticles offer a unique opportunity in the field of biocatalysis thanks to their outstanding properties. The tunable pore size in the range of mesopores allows for immobilizing bulky enzyme molecules. The large surface area improves the catalytic efficiency by increasing enzyme loading and finely dispersing the biocatalyst molecules. The easily tunable pore morphology allows for creating a proper environment to host an enzyme. The confining effect of mesopores can improve the enzyme stability and its resistance to extreme pH and temperatures. Benefits also arise from other peculiarities of nanoparticles such as Brownian motion and easy dispersion. Fossil fuel depletion and environmental pollution have led to the need for alternative sustainable and renewable energy sources such as biofuels. In this context, lignocellulosic biomass has been considered as a strategic fuel source. Cellulases are a class of hydrolytic enzymes that convert cellulose into fermentable sugars. This review is intended to survey the immobilization of cellulolytic enzymes (cellulases and β-glucosidase) onto mesoporous silica nanoparticles and their catalytic performance, with the aim to give a contribution to the urgent action required against climate change and its impacts, by biorefineries’ development.

Mesoporous silica materials offer a unique opportunity for enzyme immobilization thanks to their properties, such as tuneable pore size, large surface area and easy functionalization. However, a significant enhancement of cellulase enzyme activity entrapped inside the silica pores still represents a challenge. In this work, we immobilized cellulase by adsorption on wrinkled silica nanoparticles (WSNs), obtaining an active and stable biocatalyst. We used pentanol as co-solvent to synthesize WSNs with enhanced inter-wrinkle distance in order to improve cellulase hosting. The physical-chemical and morphological characterization of WSNs and cellulase/WSNs was performed by thermogravimetric (TG), Fourier transform infrared (FT-IR), and transmission electron microscopy (TEM) analyses. The obtained results showed that this matrix generates a favourable microenvironment for hosting cellulase. The results of the catalytic assays and operational stability confirmed the key role of size, morphology and distribution of the pores in the successful outcome of the cellulase immobilization process. The immobilization procedure used allowed preserving most of the secondary structure of the enzyme and, consequently, its catalytic activity. Moreover, the same value of glucose yield was observed for five consecutive runs, showing a high operational stability of the biocatalyst.

Candida rugosa lipase (CRL) was deposited by matrix assisted pulsed laser evaporation (MAPLE) in order to immobilize the enzyme with a preserved native conformation, which ensures its catalytic functionality. For this purpose, the composition of the MAPLE target was optimized by adding the oil phase pentane to a water solution of the amino acid 3-(3,4-dihydroxyphenyl)-2-methyl-l-alanine (m-DOPA), giving a target formed by a frozen water-lipase-pentane microemulsion. Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) were used to investigate the structure of MAPLE deposited lipase films. FTIR deconvolution of amide I band indicated a reduction of unfolding and aggregation, i.e., a better preserved lipase secondary structure in the sample deposited from the frozen microemulsion target. AFM images highlighted the absence of big aggregates on the surface of the sample. The functionality of the immobilized enzyme to promote transesterification was determined by thin layer chromatography, resulting in a modified specificity.

The enzymatic conversion of biomass-derived compounds represents a key step in the biorefinery flowsheet, allowing low-temperature high-efficiency reactions. β-Glucosidases are able to hydrolyze cellobiose into glucose. Wrinkled silica nanoparticles (WSNs) were demonstrated to be a good support for the immobilization of β-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 β-glucosidases as the active phase. Powder and structured materials were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), N₂ 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_M value showed a 20-fold decrease with respect to the batch process, due to the absence of external diffusive transport limitations.