Laccases immobilization

Gianfreda and Bollag (1994) investigated the behavior of laccase and peroxidase in the presence of a montmorillonite, a kaolinite, and a silt loam soil. They observed considerable variation in the retained activities of the two enzymes immobilized on the different supports as well as variation in the amount of each enzyme sorbed (Table 2.10). Interestingly enough, laccase immobilized on montmorillonite showed a higher specific activity (118%) than that of the free enzyme. This may be attributed to the steric modification of the immobilized enzyme or possibly due to the catalytic ability of montmorillonite itself. Their studies showed that the performance of these enzymes is significantly affected by soil mineral colloids. 

Figure 2.24. Removal of 14C-2,4-dichlorophenol by laccase immobilized on clays and soil. Reprinted from Ruggiero, P., Sarkar, J. M., and Bollag, J.-M. (1989). Detoxification of 2,4-dichlorophenol by a laccase immobihzed on soil and clay. Soil Sci. 147, 361-370, with permission from Lippincott Williams Wilkins.

Figure 16. Cu K-edge XANES taken for laccase immobilized on grafoil substrate in pH 4 sodium citrate buffer at the indicated potentials.

Y.B. (2008) Direct electrochemistry of laccase immobilized on au nanoparticles encapsulated-dendrimer bonded conducting polymer application for a catechin sensor. Analytical Chemistry, 80 (21), 8020-8027. 

Laccase immobilized by adsorption on soot promotes the process of hydroquinone oxidation by molecular oxygen. Quinone is reduced cathodically, thus accounting for a decrease in overvoltage over that for molecular oxygen reduction by about 0.3 V. 

Comparison of the exchange currents calculated per laccase molecule (/q = 3 x 10 A/laccase molecule) and per surface platinum atom (/q = 8 x 10 A/platinum atom) reveal that the activity of the enzyme is significantly higher compared to the most active catalyst for cathodic reduction of oxygen. However, the activity of immobilized laccase is about 30 times lower than that of the solubilized preparation. Therefore, it can be expected that the improvements in the method of laccase immobilization will permit its activity in the electrochemical system to be further increased. 

The adsorptional method of immobilization used in this work significantly adds to the stability of the enzyme. An electrode with laccase immobilized on soot showed stable performance for 50 hours, the polarization current being 87.5 /i A. 

Rawal, R, Chawla, S., and Pundir, C. S. (2011). Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/pol5 niline gold electrode. Anal. Biochem, 419, pp. 196-204. 

Stanescu MD, Fogorasi M, Shaskolskiy BL, Gavrilas S, Lozinsky VL (2010) New potential biocatalysts by laccase immobilization in PVA cryogel type carrier. Appl Biochem Biotechnol... 

Mousty, C., Vieille, L., Cosnier, S. Laccase immobilization in redox active layered double hydroxides A reagentless amperometric biosensor Biosens. Bioelectron. 22(8), 1733-1738 (2007). doi 10.1016/j.bios.2006.08.020... 

S. Peng, H.C. Meng, L. Zhou, J. Chang, Synthesis of novel magnetic ceUulose-chitosan composite microspheres and their apphcation in laccase immobilization. Journal of Nanoscience and Nanotechnology 14 (2014) 7010-7014. 

Rawal R, Chawla S, Devender CS, Pundir CS (2012) An amperometric biosensor based on laccase immobilized onto Fe304NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract. Enzyme Microb Technol 51(4) 179-185... 

Chawla, S Rawal, R Kumar, D. and Pundir, C.S. (2012) Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparti-cles/zinc oxide nanoparticles modified gold electrode. Anal. Biochem., 430, 16-23. 

To prepare enzyme-free ABTS ", 50 mL of 5 mM ABTS was added to 10 g of laccase immobilized on silica crystals and let stand at room temperature for approximately 7 d [46]. The oxidized ABTS solution was then separated from laccase by filtration and stored at 4°C. 

Wang, F., Guo, C., Yang, L. R,and Liu, C. Z. (2010). Magnetic mesoporous silica nano-particles Fabrication and their laccase immobilization performance. Biores. TechnoL, 101,8931-8935. 

Research revealed crosslinked chitosan microspheres to be a good carrier of immobilized enzymes. The covalent attachment of protease inhibitors to chitosan can be achieved via the primary amino groups or the hydroxyl groups of chitosan. Several researches have reported chitosan microspheres as the support to immobilize enzymes [55, 56]. For instance. Wan et al. used chitosan microspheres as the support to immobilize laccase [57]. Chitosan microspheres were added to the laccase solution in phosphate buffer. After 24 h under stirring, the laccase immobilized chitosan microspheres were obtained. Compared with the free enzyme, immobilized laccase of chitosan microspheres showed a lower specific activity but had a similar substrate affinity with improved stability which made it more attractive in the application aspect. Furthermore, they also investigated the reuse of the immobilized laccase. The results showed After being reused fifteen times, the immobilized laccase maintained at least 80% of its initial activity. 

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