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Industrial-scale applications have utilized activated carbon to immobilize enzymes. Recently, researchers identified an adsorption function of invertase hydrolysis on activated carbon in the production of enzyme nanoparticles; its key properties of high porosity, adsorption capacity, and unique surface area make it suitable for removal of various compounds. In this study, activated carbon was utilized as a support material to boost lipase catalyzed reactions in aqueous solution using deep eutectic solvent as co-solvent; effects such as carbonization temperature, impregnation ratio, carbonization time were also studied in detail.
At an active carbon formation facility, surface area and pore characteristics were determined. Surface morphology of activated carbon was examined under a scanning electron microscope (SEM), with 1000 times magnification being displayed as Figure 1. Studies of activated carbon's N2-adsorption-desorption isotherm were performed, with results showing that micropore volumes of activated carbon are conducive to creating more active sites for lipase immobilization. This time around, the micropore volume and structure of activated carbon produced by NaOH activation proved ideal for lipase adsorption. Furthermore, Guangxi people's hydrochar had an average pore width of 3.55, further supporting the hypothesis that mesoporous carbon forms during activation. Hypothesis suggests that increasing the ratio of activator to hydrochar increases its etching depth on hydrochar surface and convert more micropores to mesopores, while NaOH activation enhances development and uniformity of activated carbon surfaces. Forming an organized pore structure has now been completed, and its shape, size and structural changes in enzymes and supports are often determined by identifying and characterizing their supports. With their excellent mechanical properties and large surface area, suitable enzymes can be accommodated without excessive diffusion.
SEM image of activated carbon prepared at 1000x magnification (10um scale). [The effect of activated carbon on lipase activity can be seen].
Assay 13 activated carbon samples to test for two types of lipase enzyme, lipase and porcine lipase. Mix 1mL of free lipase sample or activated charcoal/lipase sample in one milliliter of buffer solution before centrifugng into two-milliliter centrifuge tube for analysis. Control samples utilized lipase solution prepared in phosphate buffer at a 1:1 ratio (1mL of lipase per 1mL of buffer yielded an enzyme concentration of 0.5 mg/mL), while for lipase samples in deep eutectic solvent, one mL of enzyme solution to one mL of deep eutectic solvent was used. Vortex all samples before proceeding with the next steps. Each sample was labeled A1 to A13 and then loaded with 0.1 g of activated carbon accordingly; these solutions were incubated at 350rpm thermostatic mixer for two hours. After centrifugation for 2 minutes at 8000rpm, collect the supernatant. Figure 2 depicts the relative activities of lipase and porcine lipase immobilized onto activated carbon for two hours prior to performing lipase assay. Lipase and porcine lipase controls were added for comparison. One-way analysis of variance results demonstrated an R-squared value of 0.9980 with significant differences (p0.05). This allowed selection based on highest recorded value; however, we observed no enhancement by immobilization on activated carbon, yet activity remained at around 80%; A1 and A2 produced the most red shades respectively.
(a) Relative activity of lipase after immobilization on activated carbon samples A1 to A13. Data are presented with 95% confidence intervals (error bars). (b) Relative activity of porcine lipase after immobilization on activated carbon samples A1-13.
Figure 3 depicts the effect of impregnation ratio. As indicated by its name, impregnation ratio plays a pivotal role in porosity formation and surface area development of activated carbon. Figure 3 also displays impregnation ratios between activators such as NaOH and other substances used to impregnate activated carbon (such as LiOH). At 600degC and 90 minutes carbonization time, researchers examined how impregnation ratio (IR) affects lipase immobilization at various carbonization temperatures and times. Porcine lipase activity was enhanced over other maceration ratios when tested as a control. At 400degC, the effect of activation on Teller (BET) surface area and raw material ratio of total pore volume becomes more evident; at lower activation temperatures however, ratio of activator to biomass can dramatically alter BET surface area, micropore volume, and micropore surface area. Additionally, the highest recorded level of porcine lipase activity was at 0.5 impregnation ratio (A10). When the impregnation ratio is too high, more Na molecules diffuse into pores, increasing their size and creating macropores that may inhibit enzyme immobilization and have adverse reactions during reactions. Therefore, activation plays an essential role in pore expansion by raising impregnation ratio, leading to an ever-increasing BET surface area and volume.
Effect of impregnation ratio (activated carbon A3 and A6-A9) on the relative activity of two immobilized lipases (lipase and porcine lipase) relative to their respective free enzyme counterparts.
Studies on activated carbon's effectiveness at improving lipase catalytic reaction in water are currently ongoing, and have produced excellent results in terms of immobilizing lipase and porcine lipase enzymes onto activated carbon particles. Alanine-based DES was also successfully employed as a cosolvent to enhance enzyme activity. Results have demonstrated that activated carbon can significantly enhance the enzyme activity of porcine lipase and perform superiorly than lipase itself. Findings and conclusions indicate that immobilization using activated carbon and deep eutectic solvents is an ideal approach for biotechnological applications, especially as an immobilization method. Kinetic data demonstrated that porcine lipase immobilized with activated carbon showed two and fourfold higher catalytic activity compared to pure porcine lipase and deep eutectic solvent medium, respectively. These findings and conclusions illustrate why immobilization methods employing activated carbon are ideal for biotechnological applications.
