Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

The enzyme chloroperoxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. The average pore diameter of the silica host structure (∼3 nm) was smaller than the globular CPO diameter (∼6 nm) and the enzyme remained entrapped after sol-gel maturation. The catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 g CPO per mL sol solution reached 9-12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease in cat but no changes in or . Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. The incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. The use of more acidic casting buffers (pH 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity

Solvent and Temperature Probes of the Long-Range Electron-Transfer Step in Tyramine β‑Monooxygenase: Demonstration of a Long-Range Proton-Coupled Electron-Transfer Mechanism

Tyramine β-monooxygenase (TβM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TβM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. <i>Biochemistry</i> <b>2013</b>, <i>52</i>, 1179] that the Tyr216Ala variant of TβM leads to rate-limiting ET. In this study, we present a pH–rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on <i>k</i>_cat is found in a region where <i>k</i>_cat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (<i>E</i>_a = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases

Two Poplar Hybrid Clones Differ in Phenolic Antioxidant Levels and Polyphenol Oxidase Activity in Response to High Salt and Boron Irrigation

Poplar hybrids can be used for selenium (Se) and boron (B) phytoremediation under saline conditions. The phenolic antioxidant stress response of two salt and B tolerant poplar hybrids of parentage Populus trichocarpa × <i>nigra</i> × <i>deltoides</i> was studied using high-performance liquid chromatography (HPLC) and absorption-based assays to determine the antioxidant capacity, total phenolic content, hydroxycinnamic acid levels, and the enzyme activity of l-phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), phenol peroxidase (POD), and laccase. Most remarkable was the contrasting response of the two poplar clones for PPO activity and phenolic levels to irrigation with high salt/B water. To cope with stressful growing conditions, only one clone increased its phenolic antioxidant level, and each clone displayed different PPO isoform patterns. Our study shows that poplar hybrids of the same parentage can differ in their salt/B stress coping mechanism