Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful 9 biotechnological tools. HRP is often termed the classical plant heme peroxidase, and 10 although it has been studied for decades our understanding has deepened since its 11 cloning and subsequent expression, which has enabled numerous mutational and 12 protein engineering studies. SBP, however, has been neglected until recently; despite 13 offering a real alternative to HRP that actually outperforms it in terms of stability. 14 SBP is now used in numerous biotechnological applications, including biosensors. 15Review of both is timely. This article summarises and discusses the main insights into 16 the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and 17 outlines its use in a variety of applications. It also reviews current knowledge and 18 applications to date of SBP, particularly biosensors. The final paragraphs speculate on 19 the future of plant heme-based peroxidases, with probable trends outlined and 20 explored. 21 22
The electrochemical properties of native soybean peroxidase (SBP) and of SBP modified with covalently attached ferrocene electron-transfer mediators within microcavities etched at the tip of 25 mum radius platinum microelectrodes are reported. The microcavities incorporate approximately 50 fmol of SBP. In the microcavity, native SBP undergoes a relatively fast reduction but a very slow oxidation. Ferrocene mediators have been attached to the SBP glycans (Fc-SBP) (approximately 1.5 ferrocene mediators per SBP molecule). Cyclic voltammetry reveals that these centers are capable of mediating the reduction of oxidized SBP and increase the rate of heterogeneous electron transfer between the enzyme and the electrode by >10-fold. Micromolar concentrations of H2O2 chemically oxidize the SBP and Fc-SBP systems leading to an electrocatalytic reduction at approximately -0.1 V. Successive additions of 2.5 micromol of H2O2 at a constant applied potential of -0.1 V gave a steady-state constant current of approximately 60 nA within 20 s. The steady-state current increased linearly with peroxide concentration for 2.5
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