Biomimetic green synthesis of ZnO nanoflowers using α-amylase: from antimicrobial to toxicological evaluation.

Biologically mediated synthesis of nanomaterials has emerged as an ecologically benign and biocompatible approach. Our study explores enzymatic synthesis, utilizing α-amylase to synthesize ZnO nanoflowers (ZnO-NFs). X-ray diffraction and energy-dispersive X-ray spectroscopy revealed crystal structure and elemental composition. Dynamic light scattering analysis indicates that ZnO-NFs possess a size of 101 nm. Transmission electron microscopy showed a star-shaped morphology of ZnO-NFs with petal-like structures. ZnO-NFs exhibit potent photocatalytic properties, degrading 90% eosin, 87% methylene blue, and 81% reactive red dyes under UV light, with kinetics fitting the Langmuir-Hinshelwood pseudo-first-order rate law. The impact of pH and interfering substances on dye degradation was explored. ZnO-NFs display efficient bacteriocidal activity against different Gram-positive and negative strains, antibiofilm potential (especially with P. aeruginosa), and hemocompatibility up to 600 ppm, suggesting versatile potential in healthcare and environmental remediation applications.

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.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture.

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Carbonic anhydrase as a tool to mitigate global warming.

The global average temperature breaks the record every year, and this unprecedented speed at which it is unfolding is causing serious climate change which in turn impacts the lives of humans and other living organisms. Thus, it is imperative to take immediate action to limit global warming. Increased CO2 emission from the industrial sector that relies on fossil fuels is the major culprit. Mitigating global warming is an uphill battle that involves an integration of technologies such as switching to renewable energy, increasing the carbon sink capacity, and implementing carbon capture and sequestration (CCS) on major sources of CO2 emissions. Among all these methods, CCS is globally accepted as a potential technology to address this climate change. CCS using carbonic anhydrase (CA) is gaining momentum due to its advantages over other conventional CCS technologies. CA is a metalloenzyme that catalyses a fundamental reaction for life, i.e. the interconversion of bicarbonate and protons from carbon dioxide and water. The practical application of CA requires stable CAs operating under harsh operational conditions. CAs from extremophilic microbes are the potential candidates for the sequestration of CO2 and conversion into useful by-products. The soluble free form of CA is expensive, unstable, and non-reusable in an industrial setup. Immobilization of CA on various support materials can provide a better alternative for application in the sequestration of CO2. The present review provides insight into several types of CAs, their distinctive characteristics, sources, and recent developments in CA immobilization strategies for application in CO2 sequestration.

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.

An outlook on potential protein targets of COVID-19 as a druggable site.

BACKGROUND: SARS-CoV-2 which causes COVID-19 disease has started a pandemic episode all over the world infecting millions of people and has created medical and economic crisis. From December 2019, cases originated from Wuhan city and started spreading at an alarming rate and has claimed millions of lives till now. Scientific studies suggested that this virus showed genomic similarity of about 90% with SARS-CoV and is found to be more contagious as compared to SARS-CoV and MERS-CoV. Since the pandemic, virus has undergone constant mutation and few strains have raised public concern like Delta and Omicron variants of SARS-CoV-2. OBJECTIVE: This review focuses on the structural features of SARS-CoV-2 proteins and host proteins as well as their mechanism of action. We have also elucidated the repurposed drugs that have shown potency to inhibit these protein targets in combating COVID-19. Moreover, the article discusses the vaccines approved so far and those under clinical trials for their efficacy against COVID-19. CONCLUSION: Using cryo-electron microscopy or X-ray diffraction, hundreds of crystallographic data of SARS-CoV-2 proteins have been published including structural and non-structural proteins. These proteins have a significant role at different aspects in the viral machinery and presented themselves as potential target for drug designing and therapeutic interventions. Also, there are few host cell proteins which helps in SARS-CoV-2 entry and proteolytic cleavage required for viral infection.

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.

Antibacterial efficacy of facile cyanobacterial silver nanoparticles inferred by antioxidant mechanism.

Nanoparticles (NPs) have gained importance in technological advances owing to their user friendly enhanced and efficient physical, chemical, and biological characteristics compared to their bulk counterparts. Biological synthesis of NPs by using a microorganism, enzymes, or plant extracts offers a greener and eco-friendly approach besides many advantages over physical or chemical approaches. This study reports the biosynthesis of silver nanoparticles (AgNPs) using Nostoc muscorum NCCU 442 aqueous extract as the reducing and capping agent for AgNPs synthesis. The synthesized nanoparticles were characterized by UV-VIS spectrum, SEM, EDS, TEM, AFM, DLS and XRD. Results showed distinguishing polycrystalline nature of synthesized AgNPs with surface plasmon significant band in the size range of 6-45nm with average 30 size nm. FT-IR study revealed the role of secondary metabolites present in aqueous extract for the synthesis of AgNPs. Biological activities of purified AgNPs as antioxidant and antibacterial potential showed the highest antibacterial activity against Staphylococcus aureus MTCC 902.

Immobilization of Transglutaminase on multi-walled carbon nanotubes and its application as bioinspired hydrogel scaffolds.

The chemical cross-linkers are difficult to be removed from the scaffold materials, which limit their application in tissue engineering; designing an efficient biocompatible hydrogel is though challenging is desirable. The aim of the present study was to immobilize microbial Transglutaminase (MTGase) enzyme on multi-walled carbon nanotubes (MWCNTs) for its application in hydrogel scaffolds designing. MTGase from Streptomyces mobaraensis, a non-toxic biological cross-linker, was employed for a greener approach with enhanced biochemical and structural properties. The maximum immobilization efficiency of 58% was achieved when MTGase was covalently coupled on MWCNTs. The kinetic studies showed 4.76-fold increase in catalytic efficiency and good reusability upto seven cycles. Attachment of enzyme on MWCNTs surface was studied through SEM and FTIR. The immobilized enzyme showed good cross-linking efficiency in gelatin hydrogel scaffold resulting decrease in swelling ratio of hydrogel. Our findings report for the first time the development of novel biocompatible hydrogel scaffolds with immobilized MTGase onto MWCNTS. Inevitable damage of hydrogels are incurred during their applications. To offset the damage of hydrogels, the creation of bioinspired hydrogels emulating native tissue microenvironment is highly significant. Microbial TGase holds promising future with its applicability as a cross-linker of hydrogel scaffolds in the area of tissue engineering.

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.

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.

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.

Ampicillin Silver Nanoformulations against Multidrug resistant bacteria.

The present study reported a single step synthesis of silver nanoparticles using ampicillin (Amp-AgNps), a second-generation ß lactam antibiotic to get nanoformulation having dual properties that of antibiotic and silver. The Amp-AgNps was characterized by UV-VIS spectroscopy, TEM, XRD, FTIR and TGA. FTIR and TGA results suggested that amine group of Ampicllin reduce the metalic silver into nano form. These results were further validated by computational molecular dynamics simulation. The antibacterial potential of Amp-AgNps was investigated against sensitive and drug resistant bacteria. MIC of Amp-AgNps against 6 different bacterial strains were in the range of 3-28 µg/ml which is much lower than the MIC of ampicillin (12-720 µg/ml) and chemically synthesized silver nanoparticles (280-640 µg/ml). The repeated exposure to drugs may lead to development of resistance mechanism in bacteria against that drug, so the efficacy of Amp-AgNps after repeated exposure to bacterial strains were also studied. The results indicate that bacterial strains do not show any resistance to these Amp-AgNps even after exposure up to 15 successive cycles. The biocompatibility of these Amp-AgNps was checked against cell lines by using Keratinocytes cell lines (HaCaT).

Removal of toxic contaminants from water by sustainable green synthesised non-toxic silver nanoparticles.

The study describes the synthesis of silver nanoparticles using 21 different plant extracts having medicinal properties. Molecular ultraviolet-visible spectroscopy shows that the λmax of nanoparticles synthesised by different plant extracts varied and ranged between 400 and 468 nm. The ultraviolet results revealed that although synthesis of nanoparticles occurred by all plant extracts successfully, their size varies, this was further confirmed by differential light scattering. The synthesised nanoparticles were investigated for their antimicrobial properties. The most promising silver nanoparticles Ocimum sanctum and Artemisia annua assisted were further characterised using transmission electron microscopy and energy dispersive X-ray spectroscopy (EDX). EDX data confirms that synthesised nanoparticles are highly pure. Further these two plant assisted nanoparticles were studied for chemocatalytic and adsorptive properties. The silver nanoparticles from Ocimum sanctum can catalyse the reduction of 4-nitrophenol (63%) within 20 min in the presence of NaBH4, whereas Artemisia annua assisted silver nanoparticles did not show significant chemocatalytic activity. Both the promising nanoparticles can efficiently adsorb textile dyes from aqueous solutions. These synthesised nanoparticles were also exploited to remove microbial and other contaminants from Yamuna River water. The nanoparticles show excellent antimicrobial properties and can be reused repeatedly.

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.

Reusable magnetic nanobiocatalyst for synthesis of silver and gold nanoparticles.

In the present work, we describe a simple procedure for the biosynthesis of nanosilver and gold by the reduction of silver nitrate and auric chloride respectively using a nanobiocatalyst. The nanobiocatalyst was prepared by covalent coupling of alpha amylase on (3-aminopropyl)triethoxysilane (APTES) modified iron oxide magnetic nanoparticles. The nanobiocatalyst retains 77% of its activity as compared to free alpha amylase. The nanobiocatalyst can be used up to three consecutive cycles for the synthesis of nano silver and gold. The biosynthesized nanoparticles after each cycle were characterized by UV-vis spectrophotometer, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscope (TEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Silver and gold nanoparticles of same morphology and dimensions were formed in each cycle. The procedure for synthesis of nanoparticles using an immobilized enzyme is eco-friendly and can be used repeatedly.

Screening of cyanobacterial extracts for synthesis of silver nanoparticles.

Improvement of reliable and eco-friendly process for synthesis of metallic nanoparticles is a significant step in the field of application nanotechnology. One approach that shows vast potential is based on the biosynthesis of nanoparticles using micro-organisms. In this study, biosynthesis of silver nanoparticles (AgNP) using 30 cyanobacteria were investigated. Cyanobacterial aqueous extracts were subjected to AgNP synthesis at 30 °C. Scanning of these aqueous extracts containing AgNP in UV-Visible range showed single peak. The λ max for different extracts varied and ranged between 440 and 490 nm that correspond to the "plasmon absorbance" of AgNP. Micrographs from scanning electron microscope of AgNP from cyanobacterial extracts showed that though synthesis of nanoparticles occurred in all strains but their reaction time, shape and size varied. Majority of the nanoparticles were spherical. Time taken for induction of nanoparticles synthesis by cyanobacterial extracts ranged from 30 to 360 h and their size from 38 to 88 nm. In terms of size Cylindrospermum stagnale NCCU-104 was the best organism with 38 and 40 nm. But in terms of time Microcheate sp. NCCU-342 was the best organism as it took 30 h for AgNP synthesis.

Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis.

In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy.

Alpha amylase assisted synthesis of TiO2 nanoparticles: structural characterization and application as antibacterial agents.

The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that the morphology of nanoparticles depends upon the enzyme concentration used at the time of synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was found to be 62.50 µg/ml for both the bacterial strains. The inhibition was further confirmed using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.