Tyrosinase, immobilization

Naidja et al. (1997) showed that tyrosinase immobilized on montmorillonite coated with Al hydroxide polymers retains a higher specific activity than the free... 

Wada S, Ichikawa H, Tatsumi K. Removal of phenols with tyrosinase immobilized on magnetite. Water Sci Technol 1992 26(9-11) 2057-2059. 

Sometimes it is necessary to use different substrates, cofactors and mediators in order to increase the sensitivity. Due to the irreversible nature of many analyte-enzyme interactions, sensing elements must either be reactivated or should be disposable elements. An example is the determination of diazinon and dichlorvos that uses tyrosinase immobilized on screen printed electrodes, and a redox mediator (l,2-naphtaquinone-4-sulfonate) (Everett and Rechnitz, 1998). For other systems a further drawback is the lack of selectivity of some of the enzymes involved. 

Biosensors based on a Clark oxygen electrode, coupled to tyrosinase immobilized by three different methods, were investigated for the determination of phenol in real matrices, such as water of various natural sources, industrial wastes and oil press. The feasibility study included direct use of the biosensors and in situ analysis. An integrated system, incorporating SPE, desorption, fractionation and biosensor detection, was validated for screening phenolic compounds in water. Two types of electrode were tested, solid graphite and CPE incorporating tyrosinase. Correct analyses were found for river water samples spiked with phenol (10 p.gL ), p-cresol (25 p.gL ) and catechol (1 A mul-... 

F.C. Wu, R.L. Tseng, R.S. Juang, Enhanced abilities of highly swollen chitosan beads for color removal and tyrosinase immobilization . Journal of Hazardous... 

Yin H, Zhou Y, Xu J, Ai S, Cui L, Zhu L (2010) Amperometric biosensOT based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine-siUc fibroin film and its application to determine bisphenol A. Anal Chim Acta 659 144—150... 

In another biosensor construction variant, an enzyme electrode based on tyrosinase immobilized with ordered mesoporous carbon-Au (OMC-Au), L-lysine membrane and Au nanoparticles (tyrosinase/OMC-Au/L-lysine/Au) was combined with ANNs for the simultaneous determination of catechol and hydroquinone in compost bioremediation of municipal solid waste [35]. Limits of detection achieved were below 1 pM, demonstrating this is an appropriate tool for the quantitative study of a composting system. 

Zhou YL, Tian RH, Zhi JF (2007) Amperometric biosensor based rai tyrosinase immobilized on a boron-doped diamond electrode. Biosens Bioelectron 22 822-828... 

Production of drugs from enzyme activity is seen as an interesting and selective alternative to chemical production. Production of l-DOPA by tyrosinase was particularly investigated [226]. Before immobilization of the enzyme, the support was... 

Some non-silica sol-gel materials have also been developed to immobilize bioactive molecules for the construction of biosensors and to synthesize new catalysts for the functional devices. Liu et al. [33] proved that alumina sol-gel was a suitable matrix to improve the immobilization of tyrosinase for detection of trace phenols. Titania is another kind of non-silica material easily obtained from the sol-gel process [34, 35], Luckarift et al. [36] introduced a new method for enzyme immobilization in a bio-mimetic silica support. In this biosilicification process precipitation was catalyzed by the R5 peptide, the repeat unit of the silaffin, which was identified from the diatom Cylindrotheca fusiformis. During the enzyme immobilization in biosilicification the reaction mixture consisted of silicic acid (hydrolyzed tetramethyl orthosilicate) and R5 peptide and enzyme. In the process of precipitation the reaction enzyme was entrapped and nm-sized biosilica-immobilized spheres were formed. Carturan et al. [11] developed a biosil method for the encapsulation of plant and animal cells. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

J. Yu, S. Liu, and H.X. Ju, Mediator-free phenol sensor based on titania sol-gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method. Biosens. Bioelectron. 19, 509-514 (2003). 

Duran N, Rosa MA, D Annibale A and Gianfreda L. 2002. Applications of laccases and tyrosinases (phe-noloxidases) immobilized on different supports a review. Enzyme Microb Technol 31 907-931. 

Figu re 3.3 Conceptual process model for application of a coupled tyrosinase-laccase reaction converting tyrosol. Immobilized enzymes are first characterized with respect to substrate conversion rates, using tyrosol and hydroxytyrosol as substrates for tyrosinase and laccase, respectively. One hundred percent conversion can be achieved in Reactor 1 by use of sufficient tyrosinase... 

Ruggiero et al. (1989) investigated the ability of a natural silt loam soil and the clay minerals, montmorillonite (Mte) and kaolinite (Kte), to immobilize laccase. They compared the catalytic abilities of the soil-enzyme and clay-enzyme complexes to degrade 2,4-dichlorophenol. They found that the immobilized laccase remains active in removing the substrate even after 15 repeated cycles of substrate addition (Figure 2.24). However, Claus and Filip (1988) found that the activity of tyrosinase, laccase, and peroxidase is inhibited by immobilization on bentonite. The type of saturating cations on clay surfaces also substantially influences enzymatic activity (Claus and Filip, 1990). 

A glassy C electrode, which was immobilized with tyrosinase, was used for amperometric detection of phenol in a polyimide chip. Phenol was enzymatically converted to catechol, which was then oxidized to quinone during detection. Chlorophenol can also be detected, but this is achieved after a dechlorination step (to phenol) using a Mg/Pd metal catalyst [229]. 

Gold nanoparticles have been used to immobilize micro-peroxidase 11,46 tyrosinase,47 and hemoglobin48 to construct amperometric sensors, while silver nanoparticles have been used to enhance electron transfer of cytochrome c and myoglobin onto pyrolitic graphite electrodes.49 The use of semiconductor and oxide nanoparticles has also been reported, such as horseradish peroxidase (HRP) on TiC>2 nanoparticles,50 as well as Fe304 and MnC>4 nanoparticles to immobilize and facilitate direct electron transfer.51... 

Muller and co-workers have demonstrated the potential of coupling several spontaneous chemical steps to biocatalysis in a one-pot domino reaction to form bicyclo[2.2.2]octenes.13 A tyrosinase from mushrooms was immobilized on glass beads with the phenol substrate in a mixture of chloroform and a dienophile under air. Tyrosinase can transform a wide variety of phenols to the corresponding catechol, and the presence of air resulted in spontaneous oxidation to the ortho-quinone (Scheme 21.3). The presence of a dienophile then resulted in a Diels-Alder cycloaddition to form the bicyclo[2.2.2]octene product. Significant yields were achieved with a broad range of phenols and dienophiles. 

The utilization of a DNA matrix to aid enzyme immobilization is a promising alternative for the development of new biosensors and its application in flow systems. A DNA-tyrosinase carbon paste electrode [95] showed excellent performance for detection of cathecol in a FIA system and suggests that other enzymatic biosensors can benefit from the presence of DNA. 

A conducting, polymeric film of poly(indole-5-carboxylic acid) has been employed for covalent immobilization of tyrosinase, which retains catalytic activity and catalyzes oxidation of catechol to the quinone <2006MI41>. Poly(l-vinylpyrrole), polyfl-vinylindole), and some methyl-substituted compounds of poly(l-vinylindole) are of potential interest as photorefractive materials with a relatively low glass-transition temperature and requiring a lower quantity of plasticizer in the final photorefractive blend <2001MI253>. Polymers of 5,6-dihydroxyindoles fall within the peculiar class of pigments known as eumelanins and their chemistry has been reviewed <2005AHC(89)1>. 

By co-immobilizing tyrosinase with a serine esterase on a gold electrode, it is possible to establish a multistep reaction pathway that allows the activity of the esterase to be determined indirectly via measurement of o-quinone reduction at the electrode. The molecular architecture of a bi-enzyme sensor interface is shown schematically in Figure 57.13. 

The relative sensitivity of tyrosinase-based sensors for different phenohc compounds very much depends on the electrode material, the immobilization matrix or procedure, whether a transducing chemistry is incorporated, and... 

---