Biochemical Characterization of Laccase from Spirulina CPCC-695 and Their Role in Estrone Degradation.

The addition of exogenous endocrine disrupting compounds (EDCs) like estrone, in the food chain through the aquatic system, disrupts steroid biosynthesis and metabolism by altering either the genomic or non-genomic pathway that eventually results in various diseases. Thus, bioremediation of these compounds is urgently required to prevent their addition and persistence in the environment. Enzymatic degradation has proven to be a knight in shining armour as it is safe and generates no toxic products. The multicopper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase), laccase with the potential to degrade both phenolic and non-phenolic substrates has recently gained attention. In this study, the laccase was purified, characterized, and used to study estrone degradation. The culture filtrate (crude laccase) was concentrated and precipitated using cold-acetone and dialyzed against tris buffer (50 mM) giving a four-fold partially purified form, with 45.56% yield and 204.14 U/mg as specific activity and a single peak at 250-300 nm. The partially purified laccase was approximately 80 kDa as estimated by SDS-PAGE preferred ABTS as substrate. Both crude and partially purified laccase showed maximum activity at pH 3.0, 40 °C, and 4 mM ABTS. Kinetic constants (Km, Vmax) of crude and partially purified laccase were found to be 0.83 mM; 494.31 mM/min, and 0.58 mM; 480.54 mM/min respectively. Iron sulphate and sodium azide inhibited laccase maximally. Crude and partially purified laccase degradation efficiency was 87.55 and 91.35% respectively. Spirulina CPCC-695 laccase with efficient estrone degradation ability renders them promising candidates for EDCs bioremediation.

Exogenous salicylic acid mediated herbicide (Paraquat) resistance in cyanobacterial biofertilizer Microchaete sp. NCCU-342.

Paddy field farming remains the dominant form of growing rice in modern times as the rice is the staple food for over half the world's population and is closely associated with food security and political stability of many countries. Record increase in rice production have been observed since the start of the Green Revolution. India is one of the largest paddy producer and exporter in the world. However, constant use of chemical herbicide like paraquat had shown adverse impact on the rice yield. Non-target organisms of the habitat including cyanobacterial paddy biofertilizer face the herbicide toxicity and are unable to perform efficiently their role as biofertilizer. Therefore, in the present study, an attempt has been made to enhance the paraquat resistance in rice biofertilizer (Microchaete sp. NCCU-342) by exogenous addition of salicylic acid. Paraquat showed toxicity in Microchaete sp. NCCU-342 in a dose-dependent manner. Concentration of paraquat >1.0 µM exhibited lethal effect since the beginning. Through successive narrow range experiment, LD50 value of paraquat was obtained as 0.6 µM. Biomass exposed to paraquat (LD50 value) and salicylic acid (0.3 mM) showed mitigation in free radical production (2.20 % MDA and 1.69 % H2O2) and enhancement in the activity of the antioxidant enzymes activity, i.e. SOD, CAT, APX (137.76 %, 87.45 %, 118 %, respectively) and osmolytes (3.8 % proline and 21.51% sucrose). Thus, for sustainable agricultural practice, especially for paddy field cyanobacterial biofertilizer, application of salicylic acid or organism with higher salicylic acid production ability may be an alternative to overcome the paraquat toxicity.

Biosynthesis, characterization and biomedical potential of Arthrospira indica SOSA-4 mediated SeNPs.

The use of aqueous cyanobacterial extracts for selenium nanoparticle (SeNP) synthesis is considered green, cost-effective, and eco-friendly technology that is more advanced than physical and chemical methods. In the current study, an aqueous extract of Arthrospira indica SOSA-4 was used as a reducing and stabilizing agent for the green synthesis of SeNPs. The UV-Visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-Ray diffraction, Raman spectroscopy, Atomic force microscopy (AFM), Scanning electron microscopy-Energy Dispersive X-Ray spectroscopy(SEM-EDX), and Transmission electron microscopy (TEM) were performed to characterize the biosynthesized SeNPs. Gas chromatography-Mass spectrometry (GC-MS) was also performed to know the composition of the cyanobacterial extract. SEM, TEM, and AFM showed the average size of SeNPs to be 8.5 nm, 9 nm, and 8.7 nm respectively. FT-IR analysis demonstrated the presence of functional groups on the SeNPs that acted as stabilizing agents. XRD pattern and Raman spectroscopy showed the amorphous nature of SeNPs. Synthesized SeNPs showed significant antioxidant activity in DPPH, FRAP, SOR, and ABTS assay. SeNPs showed good anti-microbial activity against Staphylococcus aureus, Escherichia coli, Candida albicans, Candida glabrata, and Candida tropicalis and good anti-cancer activity in MTT assay, Trypan assay, and Flow cytometry analysis against MCF-7, SiHa, and SW480 cell lines. Non-toxicity of SeNPs against normal cell line (HEK-293) was an additional property that affirmed its potential as a bio-compatible nanomaterial.

Ameliorative effects of endogenous and exogenous indole-3-acetic acid on atrazine stressed paddy field cyanobacterial biofertilizer Cylindrospermum stagnale.

Across the world, paddy fields naturally harbour cyanobacteria that function as biofertilizers and secrete various compounds like Indole-3-acetic acid (IAA) that help organisms in regulating their growth. Also, paddy field farming utilizes large amounts of pesticides (e.g. atrazine); but their continued application in the agricultural field causes toxicity in non-target cyanobacterial species that hinder their performance as a biofertilizer. Hence, the current study is an attempt to ameliorate the atrazine stress in cyanobacterium Cylindrospermum stagnale by addition of IAA (1 mM each) under different atrazine levels (0, 60, 80, 100, 120, 140 µg/l). Atrazine toxicity affected C. stagnale in a dose-dependent manner further experiments revealed that both the exogenous and endogenous IAA mitigated the detrimental effects of atrazine. It reduced MDA content and simultaneously increased chlorophyll content, total protein content, and multiple antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)] at 140 µg/l. A molecular docking study revealed that the pesticide binds to the D1 protein of the photoelectric chain in photosynthesis. Hence, the application of IAA or cyanobacterial biofertilizer that secretes a sufficient amount of IAA may assist sustainable agriculture in counteracting the atrazine toxicity.

Synthesis, purification and characterization of Plectonema derived AgNPs with elucidation of the role of protein in nanoparticle stabilization.

Driven by the need to biosynthesize alternate biomedical agents to prevent and treat infection, silver nanoparticles have surfaced as a promising avenue. Cyanobacteria-derived nanomaterial synthesis is of substantive interest as it offers an eco-friendly, cost-effective, sustainable, and biocompatible route for further development. In the present study optimal conditions for synthesis of silver nanoparticles (AgNPs) were 1 : 9 v/v [cell extract: AgNO3 (1 mM)], pH 7.4, and 30 °C reaction temperatures. Synthesis of nanoparticles was monitored by UV-vis spectrophotometry and the maximum absorbance was observed at a wavelength of 420 nm. SEM with EDX analysis confirmed 96.85% silver by weight which revealed the purity of AgNPs. TEM & XRD analysis exhibited a particle size of ∼12 nm with crystalline nature. FTIR analysis confirmed the presence of possible biomolecules involved in the synthesis and stabilization of AgNPs. Decapping of AgNPs followed by SDS-PAGE, LCMS and MALDI TOF analysis elucidates the proteinaceous nature of the capping and stabilizing agent. Cyanobacterial-derived capped AgNPs showed more cytotoxicicity towards a non-small cell lung cancer (A549) cell line, free radical scavenger and an antimicrobial than de-capped AgNPs. In addition they showed significant synergistic characteristics with antibiotics and fungicides. The test revealed that the capped AgNPs were biocompatible with good anti-inflammatory properties. The blend of antimicrobial and biocompatible properties, coupled with their intrinsic "green" and facile synthesis, made these biogenic nanoparticles particularly attractive for future applications in nanomedicine.

Screening, characterisation and bioactivities of green fabricated TiO2 NP via cyanobacterial extract.

The present study was aimed at exploring 37 strains of cyanobacteria for the biofabrication of TiO2 NP and evaluation of their antioxidant, antifungal, antibacterial and hemolytic activity. Screening of cyanobacterial strains was done via SEM, followed by optimisation and characterisation of the best strain. Synechocystis NCCU-370 appeared as the best strain for the synthesis of TiO2 NP in terms of size (73.39 nm) and time (24 h) after screening. Following optimisation, nanoparticles were synthesised in 12 h having an average grain size of 16 nm. The aqueous extract preparation required heating of 5 mg/ml of powdered biomass to 60 °C for 10 min. Optimum conditions for the synthesis of TiO2 NP were found to be pH 7, 30 °C and 12-h cell extract exposure to 0.1 mM of salt. Antioxidant activity was evaluated via DPPH, ABTS and FRAP assay. Antifungal potential was explored against Candida albicans (MIC = 125 µg/ml), Candida glabrata (MIC = 500 µg/ml) and Candida tropicalis (MIC = 250 µg/ml), whereas antibacterial potential was gauged for Bacillus cereus (MIC = 31.25 µg/ml), Escherichia coli (MIC = 31.25 µg/ml) and Klebsiella pneumoniae (MIC = 500 µg/ml) strains. Biogenic TiO2 NP demonstrated partial synergistic effect and excellent biocompatibility.

Mitigative effect of biosynthesized SeNPs on cyanobacteria under paraquat toxicity.

Increasing population has resulted in increased food demand. Pesticides like paraquat (PQ) have been used indiscriminately to increase the growth and yield of crops. However, this has adversely affected a wide spectrum of non-target organisms like cyanobacteria that are used as a bio-fertilizer in the rice field. In the present study, biogenic- Gloeocaspa gelatinosa NCCU -430 mediated selenium nanoparticles (SeNPs) were synthesized and characterized using different techniques including UV-Visible spectroscopy, XRD, FTIR, TEM and SEM-EDX for their use as PQ toxicity mitigator in cyanobacterial biofertilizer (Anabaena variabilis NCCU-442). Therefore, a comparative study was performed among control, PQ, SeNPs and SeNPs+PQ to check the efficacy of SeNPs in mitigation of PQ induced toxicity. Supplementation of SeNPs in PQ treated culture enhanced antioxidant enzymes activity i.e., SOD (7.55%), CAT (57.94%), APX (17.45%) and GR (14.72%) as compared to only PQ treated culture. The outcomes of the present study suggested that SeNPs can ameliorate the PQ induced stress that may be used in sustainable rice cultivation needed for filing the gap between requirement and supply.

Biomedical potential of Anabaena variabilis NCCU-441 based Selenium nanoparticles and their comparison with commercial nanoparticles.

Selenium nanoparticles (SeNPs) are gaining importance in the field of medicines due to their high surface area and unique properties than their other forms of selenium. In this study, biogenic selenium nanoparticles (B-SeNPs) were synthesized using cyanobacteria and their bioactivities (antioxidant, antimicrobial, anticancer and biocompatibility) were determined for comparison with commercially available chemically synthesized selenium nanoparticles (C-SeNPs). Color change of reaction mixture from sky blue to orange-red indicated the synthesis of biogenic SeNPs (B-SeNPs). UV-Vis spectra of the reaction mixture exhibited peak at 266 nm. During optimization, 30 °C of temperature, 24 h of time and 1:2 concentration ratio of sodium selenite and cell extract represented the best condition for SeNPs synthesis. Various functional groups and biochemical compounds present in the aqueous extract of Anabaena variabilis NCCU-441, which may have possibly influenced the reduction process of SeNPs were identified by FT-IR spectrum and GC-MS. The synthesized cyanobacterial SeNPs were orange red in color, spherical in shape, 10.8 nm in size and amorphous in nature. The B-SeNPs showed better anti-oxidant (DPPH, FRAP, SOR and ABTS assays), anti-microbial (antibacterial and antifungal) and anti-cancer activitities along with its biocompatibility in comparison to C-SeNPs suggesting higher probability of their biomedical application.

Biomedical applications of environmental friendly poly-hydroxyalkanoates.

Biological polyesters of hydroxyacids are known as polyhydroxyalkanoates (PHA). They have proved to be an alternative, environmentally friendly and attractive candidate for the replacement of petroleum-based plastics in many applications. Many bacteria synthesize these compounds as an intracellular carbon and energy compound usually under unbalanced growth conditions. Biodegradability and biocompatibility of different PHA has been studied in cell culture systems or in an animal host during the last few decades. Such investigations have proposed that PHA can be used as biomaterials for applications in conventional medical devices such as sutures, patches, meshes, implants, and tissue engineering scaffolds as well. Moreover, findings related to encapsulation capability and degradation kinetics of some PHA polymers has paved their way for development of controlled drug delivery systems. The present review discusses about bio-plastics, their characteristics, examines the key findings and recent advances highlighting the usage of bio-plastics in different medical devices. The patents concerning to PHA application in biomedical field have been also enlisted that will provide a brief overview of the status of research in bio-plastic. This would help medical researchers and practitioners to replace the synthetic plastics aids that are currently being used. Simultaneously, it could also prove to be a strong step in reducing the plastic pollution that surged abruptly due to the COVID-19 medical waste.

Facile green bio-fabricated silver nanoparticles from Microchaete infer dose-dependent antioxidant and anti-proliferative activity to mediate cellular apoptosis.

With the rapid development of nanotechnology, much has been anticipated with silver nanoparticles (AgNPs) due to their extensive industrial and commercial applications. However, it has raised concerns over environmental safety and human health effects. In this study, AgNPs were bio-fabricated using aqueous extract of Microchaete and their medical applications like antioxidant, anti-proliferative, and apoptosis were done. The biosynthesis of AgNPs was continuously followed by UV-vis spectrophotometric analysis. The physiochemical properties like shape, size, crystallinity, and polydispersity of the nanoparticles were determined by Scanning Electron Microscopy (SEM) along with EDX, Transmission Electron Microscope (TEM), Atomic Force Microscope (AFM), dynamic light scattering (DLS), and X-Ray Diffraction (XRD). Biosynthesized 7.0 nm sized AgNPs with the crystalline structure (crystalline size 4.8 nm) having a hydrodynamic diameter of 38.74 ± 2.6 nm was achieved due to the involvement of reducing agents present in the cyanobacterial extract. The IC50 values of the AgNPs were evaluated as 75 µg/ml and 79.41 µg/ml with HepG2 and MCF-7 cell lines. Different in-vitro cellular assays investigated in the present study exhibited antioxidant, anti-proliferative, and apoptotic activities. Probably delayed apoptosis in HepG2 and MCF-7 is due to better antioxidant activities of Microchaete based AgNPs.

Estrone degrading enzymes of Spirulina CPCC-695 and synthesis of bioplastic precursor as a by-product.

Estrone, a steroidal estrogen that is persistently contaminating the surface water has been classified as an endocrine disruptor and as Group-1 carcinogen by the World Health Organization. Long-term exposure to estrone-contaminated water disrupt physiology, behaviour and sexual development of living organisms that lead to many disorders. So, it has to be eliminated from our surrounding. Its biological degradation is a cost effective and eco-friendly approach. The present study targets to predict the degradation pathway and understand the role of cyanobacterial enzymes: oxidoreductases (laccase, peroxidase) and esterase in estrone degradation. Poly-ß-hydroxy butyrate (PHB) was also quantified as a by-product of estrone biodegradation. The estrone degradation pathway was predicted using EAWAG-BBD/PPS database. Spirulina CPCC-695 was grown in different concentration of estrone (20 mg/l, 50 mg/l, 100 mg/l and 200 mg/l). The culture without estrone was considered as control. The culture supernatant was used for testing laccase and esterase activity whereas the biomass was used to test peroxidase activity and quantify by-product (PHB). The enzymes showed concentration-dependent activities. Maximum enzyme activities were seen at 20 mg/l estrone. Spirulina CPCC-695 utilizes estrone as a carbon source and degrades it to produce pyruvate which forms acetyl CoA that undergo condensation, reduction and polymerization to form PHB. Maximum PHB (169 µg) was also produced at 20 mg/l as a by-product during degradation.

Cyanobacteria as a bioreactor for synthesis of silver nanoparticles-an effect of different reaction conditions on the size of nanoparticles and their dye decolorization ability.

The green synthesis of metallic nanoparticles has paved the way for improving and protecting the environment by decreasing the use of toxic chemicals and eliminating biological risks in biomedical applications. Biological synthesis of metal nanoparticles is gaining more importance due to simplicity, rapid rate of synthesis and eco-friendliness. In the present investigation cyanobacterial (Microchaete NCCU-342) cell free aqueous extract has been used for optimizing biosynthesis of silver nanoparticles (AgNP). The optimized reaction parameters for efficient synthesis of AgNP were: biomass quantity of 80 µg/ml, pH 5.5, 60 °C temperature, duration of 60 min UV light exposure and 1 mM AgNO3 concentration. AgNP was characterized by UV-Visible Spectrophotometery, Transmission Electron Microscopy and Dynamic light scattering. The smallest nanoparticles (obtained from biomass parameter were spherical, polydisperessed and in the range of 60-80 nm) were characterized further in a degradation study of azo dye methyl red. Degradation of methyl red within 2 h was more with AgNP (84.60%) as compared to cyanobacterial extract (49.80%).