Chitosan flakes-mediated diatom harvesting from natural water sources.

Diatom is a unicellular photosynthetic microalga that is found in diverse environments. These are decorated with siliceous cell walls called frustules. Diatoms have long been favoured by grazers such as microscopic protozoa and dinoflagellates. However, grazers typically remain intact in laboratory culturing and feed on diatom in culturing vessels and reducing biomass yield. The isolation and cultivation of diatoms in laboratories hamper diatoms' diversity and vast industrial potential. Chitosan, a biopolymer, has been widely used with other polyelectrolytes to flocculate various organic and inorganic colloids at acidic pH. Dissolved chitosan (acidic pH) has been used in various natural water samples and wastewater system for dewatering. However, untreated chitosan flakes have never been evaluated in a heterogeneous natural water environment. Since diatoms have silica surfaces, we tested chitosan for diatom separation and optimized chitosan concentration and other parameters to obtain grazer-free diatom starter culture from raw water. We also elucidated the mechanism for chitosan flakes-mediated diatom flocculation through adsorption kinetics and molecular dynamic simulation analysis. The results of this study are statistically optimized and validated, with a significant R2 value of 0.99 for the proposed model.

Assessing diatom distribution in Cambay Basin, Western Arabian Sea: impacts of oil spillage and chemical variables.

Freshwater and marine diatoms produce the majority of the oxygen in aquatic systems. Estimates range from 12,000 to 30,000 species, and spatial distribution varies globally. There is significant variation in diatom diversity based on geographical and environmental conditions as well as the physicochemical characteristics of the habitat. Therefore, understanding the underlying factors that contribute to changes in diatom community structures requires a comprehensive understanding of taxons. A study of diatom assemblages from the Cambay Basin, Western Arabian Sea, was conducted, particularly on oil fields. A total of 37 samples were collected; nine were from oil fields. We evaluated micro-oil spills using Fourier transform infrared (FTIR) analysis and microscopic techniques. Correlations were established through the ordination analysis of pernicious physical and chemical water variables (BOD, COD, TDS, pH, temperature, and DO), including principal component analysis (PCA). The oil field sites showed more total dissolved solids (TDS) and chemical oxygen demand (COD) than the respective marine control sites. The study does not display a cause-and-effect relationship, but we observed a positive correlation between increasing silica concentrations and diatom growth in oil fields. In contrast, high aluminium concentrations in oil fields negatively impacted the growth of diatom assemblage and abundance. When surveyed in nine oil fields, we found that Gomphonella pseudosphaerophorum and Nitzschia palea are well adapted to oil concentrations up to 40 ppm.

Linkers: A synergistic way for the synthesis of chimeric proteins.

In the cell, the protein domains are attached with the short oligopeptide, commonly known as linker peptide. Besides bridging, the linker assists in the domain-domain interaction and protein folding into the peculiar conformations. Linkers allow or control the movement of protein domains in the dynamic cellular environment. The recent advances in the recombinant DNA technology enable the construction of multiple gene constructs in an open reading frame. The express sequences can work in a cascade to cater for myriad functions. This trend has given momentum to incorporating bridge sequences (linker) that essentially separates the independent domains. According to the cellular need, the bridging partner can be spaced at a secure gap or requires attaching or interacting physically. The flexible or rigid linker can help to achieve such conformations in chimeric fusion proteins. The linker can improve solubility, proteolytic resistance and stability of such fusion proteins. Recently, linker aided protein switches and antibody-drug conjugates are gaining the attention of researchers worldwide. Here, we thoroughly reviewed the types of the linker, strategies for linker engineering and the composition of a linker.

Projecting phytochemical bacoside A anti-mucorale agent: An in-silico and in-vitro assessment.

Mucormycosis, a life-threatening fungal infection that primarily affects immunocompromised individuals.The protein family commonly observed in the fugus responsible for causing Mucormycosis. The attachment of spores to host cells surface, facilitated by a protein CotH, is a critical step for the invasion and progression of the disease. Therefore, CotH inhibitors have emerged as a promising therapeutic strategy for treating mucormycosis.This study presents a novel therapeutic target and ligand for controlling the growth of Mucorales. First, to identify potential CotH inhibitors, we surveyed a library antifungal compounds elaborated in AYUSearch database. Next, using machine learning-based algorithms we screend 20 potentials ligands, followed by structure-based molecular modelling and molecular trajectory analysis to identify the three most promising chemical constituents. In-vitro tube assays on selected Mucorales determined the minimum inhibitory concentrations (MIC) for screened chemotypes. The MIC assay revealed that Bacoside inhibits the growth and sporulation at 5 mg/ml concentrations, emerging as a probable CotH inhibitor. Further, the compound's toxicity was evaluated by adding it to the feed of C.elegans, and the finding suggests that the bacoside is reasonably safe at the studied concentration. The findings project bacoside A as a potential anti-mucorale lead compound that can be further validated with preclinical and clinical studies.

Asiaticoside A for the modulation of 1-TbAd- a potential target and ligand for extensive drug resistance Mycobacterium tuberculosis.

In nature, terpene nucleosides are relatively rare, with 1-tuberculosinyladenosine (1-TbAd) being an exclusive feature of Mycobacterium tuberculosis (Mtb). The convergence of nucleosides and terpene pathways in the Mtb complex appears to have emerged late in its evolutionary history. 1-TbAd (PDB ID: 3WQK) is a prominent chemical marker for Mtb and may contribute to its virulence-related properties when exported extracellularly. We gathered a comprehensive set of 270 phytochemicals from diverse Ayurvedic texts and treatment traditions. Subsequently, we conducted structure-based molecular docking analyses to identify compounds exhibiting the strongest binding affinity for 1-TbAd, highlighting their potential as drug candidates. These selected compounds were further subjected to an in-vitro growth inhibition assay against the reference strain Mycobacterium tuberculosis h37rv. Among the candidates, Asiaticoside A (ASA) emerged as a promising candidate from the pool of 270 compounds. To assess the impact of ASA on 1-TbAd expression, we employed a PCR-based mRNA expression assay, revealing ASA's ability to downregulate 1-TbAd expression in extensively drug-resistant MTb strains. Remarkably, the conventional drug rifampin showed no such effectiveness in our experiments. We further conducted molecular dynamic simulations to explore the interaction between ASA and 1-TbAd in a cellular-like environment, confirming the stability of their interaction. Also, we predicted ASA's stability toward causing inducing the random mutations in the target gene. With this, we propose a novel target and its modulator to treat extensively drug-resistant MTB.

Investigation on anti-Corona viral potential of Yarrow tea.

Achillea millefolium (Yarrow) is a herbaceous plant of Greek origin noted to treat pneumonia, common cold, cough, and other respiratory disorders. The flowers and leaves are the core part used to prepare herbal tea that gains the world's recognition as medicinal tea. Coronavirus disease is spreading across the globe, and numerous approaches are lodged to treat virus-induced lung inflammation. Here, we used the network pharmacology, metabolite analysis, docking and molecular simulation and MM-PBSA analysis to comprehend the biochemical basis of the health-boosting impact of Yarrow tea. Next, we performed the microscopic and dynamic light scattering (DLS) analysis of yarrow-treated ChAdOx1 nCoV-19 to evaluate the virucidal activity of the Yarrow. The present study investigates the druggability, metabolites and potential interaction of the title tea with genes associated with Covid-19-induced pathogenesis. Towards this, 1022 gene hits were obtained, 30 are mutually shared. Network Pharmacology and microarray gene expression analysis find the connection of PTGS2 in relieving the virus-induced inflammation. Yarrow constituents Luteolin may inhibit or down-regulate the Cyclooxygenase II (PTGS2), a plausible mechanism underlying the Yarrow's anti-inflammatory actions. Further, the Yarrow's virucidal activity was assessed towards Transmission Electron Microscopic (TEM). The Yarrow treated SARS-nCoV-2 cell exhibits the disintegration of the virus membrane. This work provides a scientific basis for further elucidating the mechanism underlying Achillea millefolium's antiviral and anti-inflammatory properties.

Linker-assisted engineering of chimeric xylanase-phytase for improved thermal tolerance of feed enzymes.

Biological enzymes are multifunctional macromolecules that can perform hundreds of reactions simultaneously. An enzyme must possess specific characteristics to meet industrial needs, such as stability over a wide pH and temperature range and high specific activity. A phytase and xylanase mixture is generally added to poultry feed to improve the bird's health and productivity. Despite this, animal farmers have noticed no difference in productivity, and a leading cause is the high temperature at which feed is pulverized, which inactivates enzymes. A thermo-stable enzyme system can overcome these hitches. Commonly, coatings and immobilization reduce losses caused by physical-chemical factors in feed processing and digestion. To this end, we engineered the multifunctional xylanase-phytase domains on a single polypeptide fused by a helical linker. First, the ideal linker sequence was chosen by computing each selected linker's root mean square deviation (RMSD). The selected helical linker provides sufficient structural flexibility for substrate binding and product release evaluated by molecular docking and molecular dynamic simulation studies. Furthermore, a domain-domain interaction has stabilized the bridging partners, attaining the thermal optima for xylanase and phytase at 90 °C. Even at the above-optimal temperature (100 °C), the recombinant PLX was relatively stable and retained 64.2% and 59.2% activity for xylanase and phytase, respectively, when surveyed for ten hours. So far, to this date, this is the highest degree of thermostability achieved by any recombinant phytase or xylanase.Communicated by Ramaswamy H. Sarma.

pCold-assisted expression of a thermostable xylanase from Bacillus amyloliquefaciens: cloning, expression and characterization.

The biotechnological application of bacterial xylanases requires a high thermostability, a catalytically active state for a broad pH range. The Bacillus amyloliquefaciens (MTCC 1270) xynA gene was amplified and cloned into the pCold vector and was expressed in Escherichia coli to evaluate the expressed proteins' thermostability. The pCold, compared to other similar vectors, has unique properties-including pH and temperature tolerance due to the presence of the cspA promoter. The recombinant xynA-pCold (rxynApC) showed the expression of xynA gene with a molecular weight of ~ 27 kDa, confirmed on SDS-PAGE. The rxynApC exhibits optimal activity at 70 °C and pH 8.0. The residual activity of the recombinant enzyme was 90% at pH 8.0. The thermal decomposition temperature (T d) value for the rxynApC enzyme was 93.33 °C obtained from the thermogravimetric analysis, indicating the potent stability of the cloned enzyme. The specific activity of native xylanase and rxynApC under optimal conditions was 32.35 and 105.5 U/mg, respectively. The structural model of the xynA gene was predicted using the in silico tool along with the active site (containing four important Tyr-166, Gly-7, Try-69 and Arg-112 amino acids). The predicted biophysical parameters of the in silico model were similar to the experimental results. The unique feature of the cspA promoter is that it gave a high expression of rxynApC enzyme having alkali and thermostable properties with high yield in surrogate host E. coli. Thus, the recombinant xynA gene can potentially be applied to different industrial needs by looking at its thermostability and enhanced enzyme activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03315-y.

Engineering of thermostable phytase-xylanase for hydrolysis of complex biopolymers.

Industrial processing of enzymes requires higher heating that affects the thermal stability of the enzyme and increases the production cost. In this study, xylanase-phytase (XP) fusion protein was generated via co-expression in a single vector with a cold-shock promoter, leading to improved activity at optimal pH, temperature and the thermal behaviour of the protein. Xylanase-phytase (XP) fusion and phytase proteins were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The XP fusion was thermally stable up to 124 °C, higher than phytase which was steady up to 113.5 °C. XP fusion exhibits higher stability at its thermal transition midpoint (T m) 108 °C, higher than the T m value of phytase which is 90 °C. Industrially efficient and environment-friendly proteins with low production cost and higher stability can be generated by 'fusion protein' technology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02936-z.

Phytase-Fe3O4 nanoparticles-loaded microcosms of silica for catalytic remediation of phytate-phosphorous from eutrophic water bodies.

Agriculture P management practices elevate the level of inorganic phosphates in soil that results in phosphorous (P) seepage into water-bodies. This is one of the key factors that have accelerated the menace of eutrophication. Phytic acid (phytate)-P-rich plant metabolite is infamous for its anti-nutrient activity and regularly oozing in to environment though discharge of mono-gastric animals. That has amplified the magnitudes of eutrophication. In this work, for catalysis of phytate-P, the metal-organic framework fabricated towards metal oxides (Fe3O4) and phytase in highly ordered microcosms of silica was employed. The synthesized framework was characterized through transmission electron microscopy (TEM) and nitrogen isotherm analysis. Average pore diameter of synthesized bisect oval shaped structures was measured around ≈200 nm. Herein, phytase and Fe3O4 nanoparticles were loaded to the cavities of microcosms through glutaraldehyde-mediated crosslinking. Whereas Fe3O4 nanoparticles act as nano-absorbents that adsorb P liberated from phytase-mediated catalysis of phytate. Kinetic analysis of free and loaded phytase has shown relatively small reduction in catalytic efficiency. These loaded microcosms have removed 60-80% of phytate-phosphate. The optimized process has reduced the growth of photoautotrophs by 50%. Additionally the magnet-assisted separation of loaded microcosms eased the reapplication of loaded microcosms tested for six independent instances. The primary studies conducted to evaluate the geno-toxicity of loaded microcosms have not shown any harmful effect on the process like cell division and seed germination. The efficacy of this method has evaluated towards on-field testing in Changa (Gujarat, India) lake.

A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.