Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus

40-45Tyrosinase and laccase activities were detected in the corm of Amorphophallus campanulatus after extraction with ethanol followed by ammonium sulphate precipitation (20-60%) and dialysis against 10 mM Na2HPO4 buffer at pH 7.0. Tyrosinase was found to be the predominant enzyme exhibiting mono- and di-phenolase activities, specificity for L-DOPA as substrate, optimum pH being 6.0, optimum temperature at 40ºC and Km at 1.05 mM. Laccase showed substrate specificity for p-phenylenediamine (p-PD), Km at 2.7 mM, optimum pH being 5.0 and was inactivated above 40ºC. Three isoforms of tyrosinase were detected on SDS-PAGE with apparent molecular mass ~127, 31 and 27 kDa respectively. On staining sections of A. campanulatus with L-DOPA as substrate and 3-methyl benzothiazolinone hydrazone (MBTH) for colour development, tyrosinase was detected in the intercellular spaces of the plant tissue. The cytosolic region did not show any colour indicating the absence of the enzyme

Epinephrine Biosensor Using Tyrosinase Immobilized Eggshell Membrane

Electrochemical biosensor based on immobilized tyrosinase was developed for the determination of epinephrine. The enzyme was extracted from a plant source Amorphophallus companulatus and immobilized on eggshell membrane. Tyrosinase catalyses oxidation of epinephrine to epinephrinequinone which is electrochemically reduced at -0.17 V versus Ag/AgCl (3 M KCl). The resulting current was used for epinephrine quantification. The sensor showed linearity in the range 3 x 10-5 to 3 x 10-4 M with a detection limit of 1 x 10-5 M. It has reusability upto 15 cycles and a shelf life of more than 6 months when stored at 4 o C

Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: application of ultra-microelectrode to enhance sucrose biosensor's sensitivity

We are reporting fabrication and characterization of electrochemical sucrose biosensor using ultra-microelectrode (UME) for the detection of heavy metal ions (Hg(II), Ag(I), Pb(II) and Cd(II)). The working UME, with 25 μ m diameter, was modified with invertase (INV, EC: 3.2.1.26) and glucose oxidase (GOD, EC: 1.1.3.4) entrapped in agarose-guar gum. The hydrophilic character of the agarose-guar gum composite matrix was checked by water contact angle measurement. The atomic force microscopy (AFM) images of the membranes showed proper confinement of both the enzymes during co-immobilization. The dynamic range for sucrose biosensor was achieved in the range of 1 × 10<SUP>-10</SUP> to 1 × 10<SUP>-7</SUP> M with lower detection limit 1 × 10<SUP>-10</SUP> M at ρH 5.5 with 9 cycles of reuse. The spectrophotometric and electrochemical studies showed linear relationship between concentration of heavy metal ions and degree of inhibition of invertase. The toxicity sequence for invertase using both methods was observed as Hg<SUP>2+</SUP> &gt; Pb<SUP>2+</SUP> &gt; Ag<SUP>+</SUP> &gt; Cd<SUP>2+</SUP>. The dynamic linear range for mercury using electrochemical biosensor was observed in the range of 5 × 10<SUP>-10</SUP> to 12.5 × 10<SUP>-10</SUP> M for sucrose. The lower detection limit for the fabricated biosensor was found to be 5 × 10<SUP>-10</SUP> M. The reliability of the electrochemical biosensor was conformed by testing the spike samples and the results were comparable with the conventional photometric DNSA method