Microbial Biofilm Inhibition Using Magnetic Cross-Linked Polyphenol Oxidase Aggregates.

Microbial biofilm accumulation poses a serious threat to the environment, presents significant challenges to different industries, and exhibits a large impact on public health. Since there has not been a conclusive answer found despite various efforts, the potential green and economical methods are being focused on, particularly the innovative approaches that employ biochemical agents. In the present study, we propose a bio-nanotechnological method using magnetic cross-linked polyphenol oxidase aggregates (PPO m-CLEA) for inhibition of microbial biofilm including multidrug resistant bacteria. Free PPO solution showed only 55-60% biofilm inhibition, whereas m-CLEA showed 70-75% inhibition, as confirmed through microscopic techniques. The carbohydrate and protein contents in biofilm extracellular polymeric substances (EPSs) were reduced significantly. The m-CLEA demonstrated reusability up to 5 cycles with consistent efficiency in biofilm inhibition. Computational work was also done where molecular docking of PPO with microbial proteins associated with biofilm formation was conducted, resulting in favorable binding scores and inter-residual interactions. Overall, both in vitro and in silico results suggest that PPO interferes with microbial cell attachment and EPS formation, thereby preventing biofilm colonization.

Nanozymes and nanoflower: Physiochemical properties, mechanism and biomedical applications.

Natural enzymes possess several drawbacks which limits their application in industries, wastewater remediation and biomedical field. Therefore, in recent years researchers have developed enzyme mimicking nanomaterials and enzymatic hybrid nanoflower which are alternatives of enzyme. Nanozymes and organic inorganic hybrid nanoflower have been developed which mimics natural enzymes functionalities such as diverse enzyme mimicking activities, enhanced catalytic activities, low cost, ease of preparation, stability and biocompatibility. Nanozymes include metal and metal oxide nanoparticles mimicking oxidases, peroxidases, superoxide dismutase and catalases while enzymatic and non-enzymatic biomolecules were used for preparing hybrid nanoflower. In this review nanozymes and hybrid nanoflower have been compared in terms of physiochemical properties, common synthetic routes, mechanism of action, modification, green synthesis and application in the field of disease diagnosis, imaging, environmental remediation and disease treatment. We also address the current challenges facing nanozyme and hybrid nanoflower research and the possible way to fulfil their potential in future.

Bio-imprinted magnetic cross-linked polyphenol oxidase aggregates for enhanced synthesis of L-dopa, a neurodegenerative therapeutic drug.

Bio-imprinted magnetic cross-linked enzyme aggregates (i-m-CLEAs) of polyphenol oxidase (PPO) obtained from potato peels were prepared using amino-functionalized magnetic nanoparticles. Bio-imprinting is being used to improve the catalytic efficiency and conformational stability of enzymes. For bio-imprinting, PPO was incubated with different imprint/template molecules (catechol, 4-methyl catechol and l-3,4-dihydroxy phenylalanine) before cross-linking with glutaraldehyde. CLEAs imprinted with 4-methyl catechol showed maximum activity as compared with non-bio-imprinted magnetic CLEAs (m-CLEAs). They were further characterized by scanning electron microscopy and confocal microscopy. In bio-imprinted m-CLEAs, half-life (t1/2) of PPO significantly improved (364.74 min) as compared to free PPO (43.58 min) and non-bio-imprinted m-CLEAs (266.54 min). Bio-imprinted m-CLEAs showed excellent thermal and storage stability as well as reusability. The CLEAs preparation were used for the synthesis of l-3,4-dihydroxyphenylalanine (L-dopa, a therapeutic drug to treat neurodegenerative disorder) and a remarkable increase in L-dopa yield (23.5-fold) was obtained as compared to free enzyme. A cost effective and reusable method has been described for the production of L-dopa.

Magnetic combi CLEA for inhibition of bacterial biofilm: A green approach.

In the present study different enzymes (α- amylase, trypsin, cellulase, horse-radish peroxidase and pectinex ultra clear) were studied for bacterial biofilm inhibition and Pectinex ultra clear showed best inhibition. So, m-combi-CLEA of Pectinex ultra clear was developed by cross linked enzyme aggregate (CLEA) formation on APTES (3-aminopropyltriethoxysilane) modified iron oxide nanoparticles. Different parameters were optimized and it was observed that 0.4 mg/ml of protein (containing 25 U/mg cellulase activity), 0.5 mg/ml BSA and 10 mM glutaraldehyde when incubated for 3 h gives 100% enzyme activity using ethanol as the precipitant. The CLEA formed were thermally more stable as compared to free enzyme. m-combi-CLEA of Pectinex ultra clear shows 75-78% biofilm inhibition of E. coli and S. aureus. Furthermore, m-combi-CLEA can be reused till 4 cycles with same efficiency. The carbohydrate contents of E. coli biofilm decreased from 64.629 µg to 6.23 µg and for S. aureus biofilm, it decreased from 58.46 µg to 5.52 µg when treated with m-combi CLEA in comparison to untreated biofilms. FTIR, darkfield illumination Fluorescence Microscopy, and Scanning Electron Microscopy was further used for characterization.

Development of low-cost paper-based biosensor of polyphenol oxidase for detection of phenolic contaminants in water and clinical samples.

In the present work, polyphenol oxidase (PPO) enzyme was purified from potato peel using three-phase partitioning (TPP). In this method, ammonium sulfate and t-butanol were added to precipitate the protein/enzyme from the crude aqueous extract. The PPO enzyme precipitated as an interfacial layer between the upper organic solvent phase and lower aqueous phase. Different purification parameters such as crude extract to t-butanol ratio, ammonium sulfate concentration, temperature, and pH were optimized for TPP. About 69% PPO enzyme activity was recovered in a single step of TPP with 9.2-fold purification. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of partially purified PPO enzyme showed molecular weight in the range of about 30-40 kDa. The PPO enzyme was then investigated for the fabrication of a portable, cost-effective, and disposable colorimetric paper biosensor or colorimetric "test strips" for detection of phenolic contaminants. PPO and a chromophore reagent (3-methyl-2-benzothiazolinone hydrazine) generated a range of color in the presence of phenolic compounds (catechol, phenol, p-cresol, 4-methyl catechol) within 15 min, and limit of detection was found to be 0.5 µM. The biosensor worked in a broad range of pH from 3 to 11 and showed good storage stability at 25 °C and 4 °C for 30 days with no significant loss of activity. The biosensor was also applied on environmental water and urine sample to show reliability of biosensor.

Exposure of biosynthesized nanoscale ZnO to Brassica juncea crop plant: morphological, biochemical and molecular aspects.

The present work describes the in vitro synthesis and characterization of Zinc oxide nanoparticles (ZnO NPs) using an enzyme alpha amylase, the synthesized nanoparticles were used to study their beneficial effect in the growth and development of Brassica juncea. Transmission Electron Microscope (TEM) image reveals the average size of ZnO NPs was 11 nm and X-ray powder diffraction (XRD) suggests nanoparticles were crystalline in nature. In-silico study confirmed lysine, glutamine and tyrosine present in alpha amylase enzyme, plays a crucial role in the reduction of Zinc acetate dihydrate to ZnO NPs. The biochemical parameters and oxidative enzymes of Brassica juncea were compared with ZnO NPs treated plants. The effect of ZnO NPs on the cellular expression of metal tolerant protein (BjMTP) and cation efflux transporter gene (BjCET2) was also studied. The results indicate that nanoparticles can be used as a replacement for traditional harmful chemical fertilizers.

Preparation and characterization of reusable magnetic combi-CLEA of cellulase and hemicellulase.

Cross-linked enzyme aggregate (CLEA) is a technology to overcome the limitation of enzymes for its application in chemical industries. The inability of repeated use of enzymes, stability and ease of separation from reaction mixture limits its applications. Here, magnetic combi-CLEA has been synthesised by adding amino-functionalized magnetic nanoparticles into pectinase ultra-clear (containing pectinases, xylanases and cellulases). Enzymes were precipitated on the surface of amino-functionalized magnetic nanoparticles with ethanol and cross-linked using glutaraldehyde. The structural characterization of magnetic combi-CLEA was studied by Scanning Electron Microscopy. Thermal stability was performed at 70 °C for pectinase and 80 °C for xylanase and cellulase respectively. Half-life (t1/2) of the xylanase, cellulase and pectinase in free form remarkably enhance from 84.51, 29.36, and 25.29 min respectively to 533.07, 187.29 and 147.44 min in magnetic-combi CLEA respectively. Magnetic combi-CLEA can be efficiently reused till 12th cycle after which pectinase, xylanase and cellulase retain 86.45%, 90.3% and 88.62% activity respectively. Using this CLEA preparation bioethanol concentration increases to 1.82-fold as compared to free enzyme, when simultaneous saccharification and fermentation was performed using wheat straw as the substrate. Magnetic combi-CLEA can be used for a variety of industrial applications like food processing, textile industry and bioethanol production.

Development of sustainable and reusable silver nanoparticle-coated glass for the treatment of contaminated water.

Water contaminants like pathogenic microbes and organic pollutants pose a serious threat to human and aquatic life forms; thus, there is an urgent need to develop a sustainable and affordable water treatment technology. Nanomaterials especially metal nanoparticles have extensive applications in wastewater treatment, but the recovery and aggregation of nanoparticles in solution is a major limitation. In the present work, green synthesized silver nanoparticles were covalently immobilized on a glass surface to prevent aggregation of nanoparticles and to enhance their applicability. Fourier transform infrared (FTIR) of silver nanoparticle (AgNP)-coated glass shows peaks of Si-O-Si, Si-O-C, and Ag-O at 1075 cm-1, 780 cm-1, and 608 cm-1 respectively which confirms the immobilization/conjugation of nanomaterial on glass surface. The surface morphology of immobilized AgNP was studied using scanning electron microscopy (SEM) which reveals nanoparticles are spherical and uniformly distributed on glass surface. The AgNP-coated glass was used for the removal of textile dyes in solution; the result indicates approximately 95% of textile dyes were removed after 5 h of treatment. Removal of microbial contaminants from Yamuna River was studied by optical density analysis and confirmed by fluorescence dye staining. The AgNP-coated glass was also studied for their reusability and the data indicates 50% removal of microbes up to the 5th cycle. To further enhance the applicability, the inhibition of bacterial biofilms were analyzed by dark-field illumination with a fluorescence microscope. Thus AgNP-coated glass can be used in the development of food/water storage containers and in textile industries.

A reusable multipurpose magnetic nanobiocatalyst for industrial applications.

A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70°C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (Km and Vmax) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63nm to 86nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39g/l and 1.59g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme.