Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli

Abstract

Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications.

Preparation of Silica Gel Obtained From Early Cretaceous Sidi Aich Sands (Central Tunisia)

“…The variation in size is caused by the aggregation of the silica gel particles, and contributes greatly to their inter-particle porosity. This mirrors the results found by Brahmi et al (2014) [22] for a similar size of silica gel, prepared from Algerian siliceous sands.…”

“…was then dissolved in 500 ml of distilled water at 160 °C, while being stirred for 10 min, to obtain the hydrated sodium silicate [20,22]. The nal product (silica gel) was extracted after the drop wise addition of a 2M solution of hydrochloric acid at pH 3, followed by washing with distilled water to eliminate chloride ions [21,23].…”

Layer-by-Layer Encapsulation of Herbicide-Degrading Bacteria for Improved Surface Properties and Compatibility in Soils

“…Therefore, the contaminant flux across the penetrated bacterial membrane/capsule interface is greater than that of intact bacterial membranes in free cells. This can also explain the advantage of the polyDADMAC capsules compared to chitosan and HPVP-co-S, as the high charge density and reactivity of quaternary amines is expected to promote higher membrane disruption [44].…”

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines

“…Yeom et al showed that CAH activity declined as a function of increasing chlorine levels in the municipal pool waters that were treated (89). A recent study showed that properly prepared 3-aminopropyltriethoxysilane (APTES)-encapsulated cells could withstand hypochlorite concentrations up to 10 ppm, providing evidence that this could be an effective biohybrid filter medium for treating chlorinated swimming pool and other waters containing cyanuric acid (91). The APTES-encapsulated cells also showed increased permeability, allowing for degradation rates of 29 mol/min per mg of CAH protein, rivaling purified enzyme rates.…”