A Novel Biomaterial for Decolorization of Azo Dyes: Myoglobin-Zn(II) Assisted Hybrid Nanoflowers.

Protein-inorganic hybrid nanoflowers are new multifunctional materials shown enhanced catalytic performance. Specially, they are used as catalyst and dye decolorizer via Fenton reaction. In this study, the Myoglobin-Zn (II) assisted hybrid nanoflowers (MbNFs@Zn) were fabricated by using myoglobin and zinc (II) ions in different synthesis conditions. The optimum morphology was characterized SEM, TEM, EDX, XRD, and FT-IR. The hemisphere and uniform morphology was obtained at pH 6 and 0.1 mg mL-1 . The size of MbNFs@Zn are 5-6 µm. The encapsulation yield was ∼95 %. In the presence of H2 O2 , the peroxidase mimic activity of MbNFs@Zn was spectrophotometrically investigated in the different pH values (4-9). The highest peroxidase mimic activity was found as 3.378 EU/mg at pH 4. MbNFs@Zn was exhibited 0.28 EU/mg after eight cycles. MbNFs@Zn has lost about 92 % of its activity. The usability of MbNFs@Zn for decolorization of azo dyes such as Congo red (CR), and Evans blue (EB) was researched at different times, temperatures and concentrations. The decolorization efficiency was found maximum as 92.3 % and 88.4 % for EB and CR dyes, respectively. MbNFs@Zn has perfect properties such as enhanced catalytic performance, high decolorization efficiency, stability and reusability, and can be excellent potential materials for many industrial applications.

Anti-microbial, anti-oxidant and wound healing capabilities of Aloe vera-incorporated hybrid nanoflowers.

The active ingredients of Aloe vera have attracted attention for their potential use in nanotechnology-based medical applications and biomaterial production. It has many therapeutic applications in modern world. This study used Aloe vera extract in different concentrations to synthesize Aloe vera-incorporated hybrid nanoflowers (AV-Nfs). The most uniform morphology in the nanoflowers obtained was at a concentration of 2 mL. The AV-Nfs were well characterized by scanning electron microscopy, X-ray spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction (XRD). The highest peroxidase-mimicking activity of the components was 1.488 EU/mg at 60°C and pH 6. The DPPH assay determined the antioxidant activity of the components and the MTT assay tested on CCD-1072Sk fibroblast cell line determined the effect of AV-Nfs on cell proliferation. Separate treatment of AV-Nfs with Cu3(PO4)2·3H2O significantly increased cell proliferation according to free Aloe vera and CuSO4. In vitro wound healing results showed that AV-Nfs could significantly close wounds compared to free Aloe vera. In this study, AV-Nfs showed antimicrobial activity against Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae at minimum inhibitory concentration of 625 µg/mL, suggesting that AV-Nfs may be used in wound healing applications with enhanced biological properties. AV-Nfs showed no activity against the yeast Candida albicans.

Synthesis and Characterization of Copper-Nanoflowers with the Utilization of Medicinal Plant Extracts for Enhanced Various Enzyme Inhibitory Activities.

In this study, organic-inorganic hybrid nanoflowers were synthesized using methanolic extracts of the medicinal plants Ajuga chamaepitys subsp. chia var. chia, Achillea wilhelmsii, Bongardia chrysogonum, Malva sylvestris, Phlomis grandiflora var. grandiflora, Verbascum sp. together with copper ions (Cu2+ ). The synthesized plant extract based-inorganic hybrid nanoflowers (PE-ihNFs) of A. chamaepitys subsp. chia var. chia (Ac-ihNFs), A. wilhelmsii (Aw-ihNfs), B. chrysogonum (Bc-ihNFs), M. sylvestris (Ms-ihNFs) P. grandiflora var. grandiflora (Pg-ihNFs), and Verbascum sp. (Vs-ihNFs) were characterized by Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray (EDX), Fourier transform infrared spectrometry (FTIR), and X-Ray Diffraction (XRD). Also, several enzymes were selected to evaluate the enzyme inhibition activities of the synthesized PE-ihNFs. For the first-time, enzymes, tyrosinase, α-amylase and α-glucosidase, acetyl and butyryl cholinesterase inhibition activities of the PE-ihNFs with comparison to their plain plant extracts were evaluated in vitro. Results show that the among all the analyzed PE-ihNFs, demonstrated better α-glucosidase & α-amylase enzyme inhibition activity compared to the plain extracts. These initial studies are promising for the synthesis of these hybrid nanoflowers containing medicinal plant extracts, which might have commercial applications in the pharmaceutical and dermo-cosmetics industries.

Hemoglobin-Inorganic Hybrid Nanoflowers with Different Metal Ions as Potential Oxygen Carrying Systems.

Protein-inorganic hybrid nanoflowers have tremendous potential in bionanotechnology due to their simple method of preparation, high stability and superior properties. Considering these features, the present study was designed to investigate the artificial blood substitution potentials of hemoglobin-inorganic hybrid nanoflowers. In this context, hemoglobin-inorganic hybrid nanoflowers (Cu-NF, Co-NF and Zn-NF) were synthesized using with different metal ions (copper, cobalt and zinc), then their oxygen carrying capacity, the hemolytic studies, in vitro oxidant/antioxidant capacity levels and oxidative stress index were reported for the first time. The present findings have revealed that Zn-NF had significant oxygen content and artificial oxygen carriers (AOC), as well as a significantly low percent hemolysis rate and a safe standard value. Also, hemolysis rate decreased along with the increases in hemoglobin content coupled with increments in nanoflower concentrations. The percentage hemolysis rate was lower than all nanoflowers at low free hemoglobin concentration, but hemolysis rates also increased with increments in concentration. The results showed that in general, Zn-NF stands out with its high total antioxidant capacity and low total oxidant capacity and oxidative stress index. The obtained results showed that Cu-NF and Co-NF, especially Zn-NF might be considered as a potential superior artificial oxygen carrier. Therefore, this nanoflower system might be act as an efficient material as a blood substitute in the near future.

Evaluating the activity and stability of sonochemically produced hemoglobin-copper hybrid nanoflowers against some metallic ions, organic solvents, and inhibitors.

The disadvantage of the conventional protein-inorganic hybrid nanoflower production method is the long incubation period of the synthesis method. This period is not suitable for practical industrial use. Herein, protein-inorganic hybrid nanoflowers were synthesized using hemoglobin and copper ion by fast sonication method for 10 min. The synthesized nanoflowers were characterized via scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, and Fouirer-transform infrared spectroscopy. The activity and stability of the nanoflowers in the presence of different metal ions, organic solvents, inhibitors, and storage conditions were also evaluated by comparing with free hemoglobin. According to obtained results, the optimum pH and temperatures of both hybrid nanoflower and free hemoglobin were pH 5 and 40 °C, respectively. At all pH levels, nanoflower was more stable than free protein and it was also more stable than the free hemoglobin at temperatures ranging between 50 °C and 80 °C. The free protein lost more than half of its activity in the presence of acetone, benzene, and N,N-dimethylformamide, while the hybrid nanoflower retained more than 70% of its activity for 2 h at 40 °C. The hybrid nanoflower activity was essentially increased in the presence of Ca2+, Zn2+, Fe2+, Cu2+ and Ni2+ (132%, 161%, 175%, 185% and 106%, respectively) at 5 mM concentration. The nanoflower retained more than 85% of its initial activity in the presence of all inhibitors. In addition, it retained all its activity for 3 days under different storage conditions, unlike free hemoglobin. The results demonstrated that new hybrid nanoflowers may be promising in different biotechnological applications such as catalytic biosensors and environmental or industrial catalytic processes.

Evaluation of organic-inorganic hybrid nanoflower's enzymatic activity in the presence of different metal ions and organic solvents.

In the present study, lipase-inorganic hybrid nanoflowers (L-hNFs) have indicated to possess several novel characteristics that are significant in terms of industrial worth. In detail, we reported the production of lipase-inorganic hybrid nanoflowers in different parameters and evaluated synthesized nanoflowers' enzymatic activity in the presence of different concentrations of metal ions and organic solvents. The optimum pHs of free enzyme, L-hNFs (0.02, 0.05, and 0.1) and L-hNFs (0.03) were pH 8, 7 and 6, respectively. L-hNFs (0.05) displayed the highest enzymatic activity in high alkaline pH values (10, 11, and 12) compared to both free enzyme and other L-hNFs. While the free enzyme showed no activity at pH 12, L-hNFs (0.05) maintained 57% of their activity. The optimum temperatures of the free lipase and L-hNFs were 30 °C and 40 °C, respectively. Free lipase activity decreased with temperature while L-hNFs had higher enzyme activity at 40 °C and 50 °C. Even in the case of increasing incubation and organic solvent and metallic ion concentration in which free lipase cannot completely maintain its activity, L-hNFs were able to protect their activity. L-hNFs exhibited excellent thermal and pH stability and high resistance to metallic ions and organic solvents at various concentrations for 3 h, 6 h, and overnight. This new lipase-inorganic hybrid nanobiocatalyst may be promising in many industrial processes such as chemical, biochemical, pharmaceutical, and biotechnological ones.

Proteinase K hybrid nanoflowers (P-hNFs) as a novel nanobiocatalytic detergent additive.

In this study, enzyme-inorganic hybrid nanoflowers were synthesized using proteinase K and Cu2+ ions. The synthesized proteinase K-Cu2+ hybrid nanoflowers (P-hNFs) were characterized by their morphology and chemical point of view by using different techniques such as SEM, FTIR, EDX, and XRD. The proteolytic activities and some important characteristics such as optimum pH and temperature of the P-hNFs were also evaluated by comparison with free proteinase K. Optimum pH values of free proteinase K and P-hNFs were determined as pH 10 and pH 11, respectively. Optimum temperatures recorded for both free proteinase K (at pH 10) and P-hNFs (at pH 11) were 40 °C. In our study, for the first time, using some commercial detergents and surfactants, the utility of the P-hNFs as a detergent additive was also systematically evaluated. In these studies, the P-hNFs exhibited better activity than free proteinase K in the presence of all surfactants (CHAPS, DOC, SDS, Triton X-100 and Tergitol) except for Tween 80. Importantly, the P-hNFs was more stable and compatible with all tested solid laundry detergents. The findings demonstrated that the P-hNFs could potentially be used as an additive in detergent formulations.

Egg white hybrid nanoflower (EW-hNF) with biomimetic polyphenol oxidase reactivity: Synthesis, characterization and potential use in decolorization of synthetic dyes.

In this study, for the first time, we described organic-inorganic hybrid nanoflowers using crude egg white as the organic component and copper (II) ions as the inorganic component under the mild conditions. The synthesized egg white-inorganic hybrid nanoflowers (EW-hNFs) were characterized using SEM, EDX, XRD and FTIR analysis. The biomimetic Polyphenol/Peroxidase like activities of synthesized egg white-inorganic hybrid nanoflowers (EW-hNFs) were determined by using various phenolics with or without H2O2. Optimum pH and temperature, kinetic parameters, reusability, pH and thermal stability of EW-hNFs were also studied. The most noteworthy aspect of our study is that synthesized EW-hNFs which consist of only egg white proteins, showed polyphenol oxidase activity. Furthermore, potential use of the EW-hNFs in the discoloration of the some synthetic dyes was also evaluated.

Self assembled snowball-like hybrid nanostructures comprising Viburnum opulus L. extract and metal ions for antimicrobial and catalytic applications.

Herein, we report the production of novel organic-inorganic nanobio-catalytic and antimicrobial agents called "nanosnowball" (NSBs) with a rational design and elucidate the increase in the catalytic and antimicrobial activities. The NSBs resemble to guelder rose were formed of the extract of Viburnum opulus (VO) as the organic component and copper (II) ions (Cu2+) as the inorganic component. The effects of the VO extract and Cu2+ concentrations on the morphology of NSBs were systematically examined and characterized with several techniques such as SEM, FT-IR, EDX and XRD. Our results demonstrated that the presence of CuO bonds in NSBs could be indication of VO extract-Cu2+ complexes. Interestingly, the NSBs exhibited peroxidase-like activity towards guaiacol used as a model substrate depending on Fenton-like reaction. While free VO extract did not show antimicrobial activity at indicated concentration (2000-125µg/mL), the NSBs showed effective antimicrobial activity against bacterial (Escherichia coli ATCC 35218, Salmonella typhi ATCC 14028, Enterococcus faecium ATCC 8459, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 25923, except Pseudomonas aeruginosa ATCC 27853 and Haemophilus influenza ATCC 49247) and fungal pathogens (Candida albicans ATCC 10231, C. glabrata ATCC 90030), respectively.

A hierarchical assembly of flower-like hybrid Turkish black radish peroxidase-Cu2+ nanobiocatalyst and its effective use in dye decolorization.

Effective dye decolorization in wastewater still shows a big challenge. Although the biological methods, especially using enzymes, offer alternative and effective process for dye degradation and overcome the limitations of chemical and physical methods such as the instability, lack of reusability and high cost of free enzymes strictly, which limit their use in many scientific and technical applications. Enzymes rapidly lose their activities in aqueous solutions and against environmental changes due to their very susceptibility and unfavorable conformations. Herein, we report preparation of the enzyme-inorganic hybrid nanostructures with flower-like shape consisting of Turkish black radish peroxidase and Cu2+ metal ions using an encouraging enzyme immobilization approach. The peroxidase-Cu2+ hybrid nanoflowers (NFs) exhibited enhanced stability and activity towards various pH values and provided excellent dye decolorization efficiency for Victoria blue (VB) dye with more than 90% within 1 h. The NFs were also repeatedly used in efficient and caused 77% VB decolorization efficiency even at tenth cycles. However, to the best of our knowledge, for the first time, we prepared peroxidase enzyme isolated from Turkish black radish incorporated NFs and used them for dye decolorization. We believe that the NFs can be promising materials for dye decolorization in real wastewater treatment.

The Effect of Pelargonium endlicherianum Fenzl. root extracts on formation of nanoparticles and their antimicrobial activities.

Herein, we report the biosynthesis of Ag NPs, for the first time, using identified antimicrobial molecules (gallic acid+apocynin) and (gallic acid+apocynin+quercetin) from the medicinal plant Pelargonium endlicherianum Fenzl. and dramatically enhanced antimicrobial activity. We also investigate the role of each molecule on formation Ag NPs and explain the increase in the antimicrobial activity of identified molecules mediated Ag NPs. The extraction protocols, 11% ethanol and 70% methanol, resulted in identification of different constituents of gallic acid+apocynin (M1) and gallic acid+apocynin+quercetin (M2) with respective concentrations. The M1-Ag and M2-Ag NPs exhibit excellent inhibitory activities towards Gram negative bacteria; Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Gram positive bacteria; Staphylococcus epidermidis ATCC 3699 bacterial using in vitro microdilution method. The minimum inhibitory concentration (MIC) values of M1-Ag and M2-Ag NPs were determined to be 7.81 and 6.25ppm for S. epidermidis, respectively. Surprisingly, MIC value for both Ag NPs was indicated to be identical as 9. 37ppm for P. aeruginosa and E., coli.

A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability.

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.

Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties.

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu(2+)) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02mgmL(-1) urease in 10mM PBS (pH 7.4) at +4°C) exhibited the highest catalytic activity of ∼2000% and ∼4000% when stored at +4°C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4°C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.

Preparation of lactoperoxidase incorporated hybrid nanoflower and its excellent activity and stability.

We report a green approach to synthesize lactoperoxidase (LPO) enzyme and metal ions hybrid nanoflowers (HNFs) and investigate mechanism underlying formation and enhanced catalytic activity and stability under different experimental parameters. The HNFs formed of LPO enzyme purified from bovine milk and copper ions (Cu(2+)) were synthesized at two different temperatures (+4 °C and 20 °C) in PBS (pH 7.4). The effects of experimental conditions, pH and storage temperatures, on the activity and stability of LPO-copper phosphate HNFs were evaluated using guaiacol as a substrate in the presence of hydrogen peroxide (H2O2). Optimum pHs were determined as pH 8 and pH 6 for LPO-copper phosphate HNF and free LPO, respectively. LPO-copper phosphate HNF has higher activity than free LPO at each pHs. Activities of LPO-copper phosphate HNF at pH 6 and pH 8 were calculated as 70.48 EU/mg, 107.23 EU/mg, respectively while free LPO shows 45.78 EU/mg and 30.12 EU/mg, respectively. Compared with free LPO, LPO-copper phosphate HNFs exhibited ∼160% and ∼360% increase in activities at pH 6 and pH 8, respectively. Additionally, LPO-copper phosphate HNFs displayed perfect reusability after six cycles. Finally, we demonstrated that LPO-copper phosphate HNFs can be utilized as a nanosensor for detection of dopamine and epinephrine.