Direct electrochemistry and bioelectrocatalysis of H2O2 reduction of recombinant tobacco peroxidase on graphite. Effect of peroxidase single-point mutation on Ca2+-modulated catalytic activity

Glutamic acid-141: a heme ‘bodyguard’ in anionic tobacco peroxidase

“…Analogous aggregates have never been observed for the recombinant HRP C (Gazaryan et al, 1994). We suppose that the aggregated fraction results from the specific interaction of tobacco peroxidase with Ca 2q that was previously reported for the native enzyme (Munteanu et al, 2000) and observed for the recombinant enzymes in electrochemical studies (Castillo et al, 2006). The latter showed that the mutation introduced partially rescued the enzyme from Ca 2q dependence, but reduced the enzyme operational stability (Castillo et al, 2006).…”

“…We suppose that the aggregated fraction results from the specific interaction of tobacco peroxidase with Ca 2q that was previously reported for the native enzyme (Munteanu et al, 2000) and observed for the recombinant enzymes in electrochemical studies (Castillo et al, 2006). The latter showed that the mutation introduced partially rescued the enzyme from Ca 2q dependence, but reduced the enzyme operational stability (Castillo et al, 2006). The decrease in calcium concentration in the course of wild-type tobacco enzyme refolding allowed us to minimize the formation of these aggregates and increase the reactivation yield to 20% (data not shown).…”

“…Electrochemical studies (Castillo et al, 2006) demonstrated lower stability of the mutant enzyme in the regime of repeated measurements, confirming the lower stability of the enzyme holoform. The native TOP showed no decrease in RZ for 6-month storage; however, the mutant exhibited a substantial decrease in RZ down to 2 and lower, i.e., the mutant enzyme loses heme much more easily than the wild-type TOP.…”

Bean sprout peroxidase biosensor based on l-cysteine self-assembled monolayer for the determination of dopamine

“…A homogenate of plant tissue materials represents not only attractive analytical properties, but also simplicity, stability, longer lifetime, low cost and reduced co-factor requirements [11][12][13]. In analytical applications immobilized enzymes are used in the development of enzymatic electrodes for the specific detection/conversion of relevant species in the food, beverage, chemical and pharmaceutical industries, as well as in agricultural analysis and environmental control [14].…”

Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode

“…6 This mechanism is expected to apply also for anionic tobacco peroxidase (TOP), which was early recognized as a promising building block for biosensors due to its high stability and substrate specificity. [7][8][9] A variety of strategies for immobilization of tobacco peroxidase have been reported, including physisorption on solid substrates, such as bare gold 10 and graphite [11][12][13][14] and on selfassembled thiol monolayers 15 or, alternatively, it has been embedded into a redox-active polymer gel. 16 In this work we show how the simultaneous incubation and deposition of a mixture of TOP and the crossing coupling agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS), which is likely to produce an extensive intra-and inter-protein coupling, results in a significant enhancement of the electrocatalytic hydrogen peroxide reduction and an overall improvement of the biosensor performance.…”