Rangasamy Savitha

β-glucosidase enzymes play a pivotal role in the hydrolysis of glycosidic bonds, with bio-catalytic applications in glycoside synthesis, lignocellulosic biomass degradation, and the processing of glycosides. This review explores the engineering of β-glucosidase mutants to enhance their cata-lytic efficiency, substrate specificity, and stability for biocatalytic applications. It also considers hy-drolytic applications of glucosidases, such as lignocellulosic biomass valorisation, as well as syn-thetic applications via reverse hydrolysis, including the production of alkyl glycosides. Additionally, it examines rational design-led mutagenesis and directed evolution strategies to tailor enzyme activity, along with structural insights gained from molecular dynamics simulations and mo-lecular docking studies. These structural insights provide an understanding of the active-site archi-tecture, catalytic mechanism, and enzyme-substrate interactions, which are useful for the rational design of improved glucosidase variants. This review underscores the synergistic role of mutagenesis studies and structural analysis in optimizing glucosidases for efficient and sustainable biocatalytic applications, paving the way for innovative applications in green chemistry.

Candida antarctica lipase B (CALB) is widely used in biocatalysis with applications in plastics degradation and chemical synthesis. CALB is activated by hydrophobic matrices and, enigmatically, shows striking activation in polar, choline-based, Deep Eutectic Solvents (DES). Herein, we show that CALB activation and stabilisation by TAAs is caused by binding to choline’s tetraalkylammonium (TAA) moiety. Several related TAA salts also caused CALB activation which was proportional to the hydrophobicity of their alkyl substituents. Remarkably, tetraoctylammonium bromide showed activation of ∼500% even at low micromolar levels. These TAA salts represent a new class of enzyme activator. Molecular modelling identified the alkylammonium binding location as a hydrophobic patch centred around Asp-145 of CALB. Binding at this site explains lipase activation in polar DES solvents and its relationship to other pathways of CALB activation. Herein, we also demonstrate that CALB, like many lipases, is activated by calcium. Intriguingly, mixed soluble activator experiments showed that calcium and choline bind to different CALB sites, suggesting a two-site model for CALB activation. These observations, along with previous findings, show that TAA activation is a widespread property of enzymes and constitutes a novel and potent means to enhance enzyme turnover and stability. Highlights: CALB is activated by choline Several tetraalkylammonium salts cause activation of CALB Hyperactivation of CALB (5-fold) by tetraoctylammonium ions occurs at low micromolar concentrations. Two independent sites for CALB activation, by calcium and TAA ions, are identified Activation at the choline binding site stabilises CALB while calcium binding destabilises the enzyme A soluble activator is demonstrated, that can be used to probe the activation mechanism of CALB or other enzymes.

Plastics play a crucial role in modern life, but their accumulation poses a serious threat to both the environment and human health. Due to their effects on the terrestrial and aquatic environment, it is essential to develop sustainable approaches to dispose of waste plastics. Traditional methods of plastic disposal, such as burning and landfilling, are problematic since they produce hazardous byproducts. Biodegradation is a potentially effective, eco-friendly approach which uses microbial consortia or isolated enzymes to break down plastic waste. Enzymes interact with plastic surfaces and hydrolyse the large polymer chains into smaller units. These byproducts can then be utilised as carbon sources by microbes, which are eventually converted into CO₂ and water. This review explores the principal approaches to plastic degradation, with a focus on existing and emerging polymers made to be readily biodegradable. In addition, sustainable valorisation methods for converting plastic waste into valuable byproducts are considered. The implementation of a circular plastic economy is expected to lead to further development, including scaling up of efficient plastic bio-upcycling processes, which can serve to stimulate environmental waste removal and value-added use of post-consumer plastic streams.

In this work, a microfluidic paper-based analytical device (μPAD) was developed to detect the biopesticide azadirachtin (Aza) through a colorimetric assay. High precision estimation of Aza is classically carried out using high performance liquid chromatography (HPLC), which requires highly skilled personnel. Acidified vanillin is a commonly used colorimetric indicator in thin layer chromatography for detection of various phytochemicals. However, the assay involves concentrated acid, which limits the choice of paper substrates for paper-based sensors and raises safety concerns. In this work, we show how the assay can be extended from the liquid phase to a paper substrate. Glass microfiber (GMF) filter paper was found to be suitable paper as it was acid resistant; besides, its hydrophilicity enabled smooth flow of reagents. A microfluidic paper-based sensor (μPAD) was developed by sandwiching 5 mm sized GMF dots between two parafilm sheets. We demonstrate the use of colorimetric assay on the μPAD for on-site detection of Aza in neem kernels. The magenta color developed upon the reaction of acidified vanillin with Aza was captured using a smart-phone and analysed using RGB levels in the image. Calibration was established using neem kernel extract of known concentration. Linearity was seen in the concentration range of 5 to 25 mg L⁻¹ Aza. A limit of detection of 2.3 mg L⁻¹ was obtained using this method. The colorimetric assay showed a relative recovery of >85% when compared with the values obtained from HPLC. The stability of the reagents on the GMF sensor was investigated to understand the storage conditions and shelf life of the reagents and sensor. The present work demonstrates the development of a portable sensor for on-site detection of phytochemicals that can be an integral part of the agricultural supply chain.

In this work, adsorption of triclosan (TCS) on nylon 66 membrane is explored to develop a preconcentration and sensing platform. Nylon 66 membrane exhibits superior sorption ability even for trace amounts of TCS (10 μg/L). Investigating the surface adsorption chemistry by XPS analysis revealed the formation of a hydrogen bond between the hydroxyl group of TCS and the amide group of nylon 66. In the absence of TCS, the amphiprotic water molecule forms a multilayer OH group on the membrane surface. However, TCS showed preferential adsorption on the membrane-replacing water molecule due to its higher hydrophobic partition coefficient. We validated the effective preconcentration of TCS on the membrane using LC-MS analysis. Performing colorimetry directly on the TCS-enriched membrane surface showed a visible color change for concentrations as low as 10 μg/L. The relative blue intensity was found to vary linearly over a concentration range of 10-100 μg/L, and we achieved a detection limit of 7 μg/L for a 5 mL sample. This method utilizes easy-to-use resources which drastically reduce the cost and complexity of analysis.