Pushplata Tripathi

Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides.

Polyacrylonitrile nanofibrous membranes (PANNFM) were prepared by electrospinning from 10 wt.% of PAN solution and its surface was modified by amidination reaction. A new chitosan degrading enzyme from Aspergillus sp. was covalently immobilized on PANNFM. Immobilization efficiency of 80% was achieved by activating PANNFM surface for 30 min followed by 2 h treatment with enzyme solution. The optimum temperature and pH for immobilized enzyme were 50°C and 5.8, respectively. The immobilized chitosanase retained >70% activity after ten repeated batch reaction and could be stored up to 60 days at 4°C with minor loss in activity. Chitosan hydrolysis using different substrates were studied using immobilized chitosanase in batch conditions. Continuous selective production of chitooligosaccharides (dimer to hexamer) by changing the temperature was achieved by PANNFM-chitosanase.

Development and characterization of nickel accumulating mutants of Aspergillus nidulans

Stable mutants of Aspergillus nidulans, resistant to 1 mM Ni were developed by step-by-step repeated culturing of the fungus on the medium containing increasing concentrations of nickel chloride. Characterization of mutants could differentiate them into two categories NiR I and NiR II. Each category of mutants exhibited alterations in growth, conidial germination and melanin secretion both in Ni-free and Ni-containing media. NiR II mutants were little slow in growth with sparse mycelia and conidiation but showed high melanin secretion and higher Ni-uptake in comparison to NiR I mutant. Studies involving metabolic and translational inhibitors could prove that Ni-accumulation was biphasic. The initial energy independent surface accumulation was found to be followed by energy dependent intarcellular uptake. Increase in the concentration of the metal in the medium or the time of exposure did not proportionately increase the metal uptake by the mutants. Ni-uptake followed Michaelis-Menton saturation kinetics, which was enhanced under optimum pH of 6.5–7.5 and reduced complexity of the medium due to free availability of ions. Resistance to Ni was found to be constitutive in NiRI mutant, and could be induced in NiRII mutant.

Mechanism to combat cobalt toxicity in cobalt resistant mutants of Aspergillus nidulans

Characteristics of fungal species tolerant to high levels of metals in natural environment can be amplified by isolation and selection of resistant mutants. Step-by-step culturing led to identification of highly stable Co-resistant (CoR) mutants of A. nidulans. Based on two distinct morphological features, Co-resistant mutants were categorized as CoRI and CoRII. The two mutants varied in their growth behavior and colony morphology that were reflected in supplemented as well as unsupplemented growth media over the generations. As compared to the CoRI, CoRII mutant exhibited sparse mycelia and conidiation but secreted higher amount of melanin. CoR mutants could tolerate up to 2.5mM Co in the medium, however, required a threshold concentration of 0.25mM Co for optimal growth and germination. Absence of Co in the medium caused a stressful situation for the CoR mutants and led to the secretion of a white extracellular precipitate found to be a glycoprotein. In response to interactions with Co-ions, CoR mutants produced oxalic acid and bioprecipitated Co as Co-oxalate providing scope for metal reclamation as well as oxalic acid extraction. The mutants could help to recover the insoluble Co-oxalate salt from aqueous solutions by entrapping it in their growing mycelial meshwork.

Recent Progress in Chitosanase Production of Monomer-Free Chitooligosaccharides: Bioprocess Strategies and Future Applications

Biological activities of chitosan oligosaccharides (COS) are well documented, and numerous reports of COS production using specific and non-specific enzymes are available. However, strategies for improving the overall yield by making it monomer free need to be developed. Continuous enzymatic production from chitosan derived from marine wastes is desirable and is cost-effective. Isolation of potential microbes showing chitosanase activity from various ecological niches, gene cloning, enzyme immobilization, and fractionation/purification of COS are some areas, where lot of work is in progress. This review covers recent measures to improve monomer-free COS production using chitosanase/non-specific enzymes and purification/fractionation of these molecules using ultrafiltration and column chromatographic techniques. Various bioprocess strategies, gene cloning for enhanced chitosanase enzyme production, and other measures for COS yield improvements have also been covered in this review. COS derivative preparation as well as COS-coated nanoparticles for efficient drug delivery are being focused in recent studies.

Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds.

Ecological samples rich in microbial diversity like cow dung, legume rhizosphere, fish waste and garden soil were used for isolation of chitosan-degrading microorganisms. Selected isolates were used for production of chitosanaseand food related bioactive compounds by conversion of biowaste. Production of glucosamine (Gln), N-acetylglucosamine (NAG), chitooligosaccharides (COS), antioxidants, antibacterial compounds and prebiotics was carried out by microbial fermentation of biowaste. The highest chitosanase activity (8 U/mL) was observed in Aspergillus sp. isolated from fish market waste and it could produce Gln and NAG while Streptomyces sp. isolated from garden soil was able to produce COS along with Gln and NAG. Radical scavenging activity was observed in culture supernatants of 35% of studied isolates, and 20% isolates secreted compounds which showed positive effect on growth of Bifidobacterium. Antibacterial compounds were produced by 40% of selected isolates and culture supernatants of two microbial isolates, Streptomyces zaomyceticus C6 and one of garden soil isolates, were effective against both gram positive and negative bacteria.

Evaluation of the Larvicidal Efficacy of Five Indigenous Weeds against an Indian Strain of Dengue Vector, Aedes aegypti L. (Diptera: Culicidae)

Background and Objectives. Aedes aegypti, dengue fever mosquito, is primarily associated with the transmission of dengue and chikungunya in tropical and subtropical regions of the world. The present investigations were carried out to assess the larvicidal efficiency of five indigenous weeds against Ae. aegypti. Methods. The 1,000 ppm hexane and ethanol extracts prepared from the leaves and stem of five plants (Achyranthes aspera, Cassia occidentalis, Catharanthus roseus, Lantana camara, and Xanthium strumarium) were screened for their larvicidal activity against early fourth instars of dengue vector. The extracts which could cause 80–100% mortality were further investigated for their efficacy. Results. The preliminary screening established the efficacy of hexane extracts as compared to the ethanol extracts. Further investigations revealed the highest larvicidal potential of A. aspera extracts exhibiting LC50 value of 82.555 ppm and 68.133 ppm, respectively. Further, their leaf extracts showed 5–85.9% higher larvicidal activity and stem extracts exhibited 0.23- to 0.85-fold more efficiency than the other four extracts. Conclusion. The present investigations suggest the possible use of A. aspera as an ideal ecofriendly, larvicidal agent for the control of dengue vector, Ae. aegypti. Future studies are, however, required to explore and identify the bioactive component involved and its mode of action.

Production, purification and characterization of a new chitosanase enzyme and improvement of chitosan pentamer and hexamer yield in an enzyme membrane reactor

Abstract An extracellular endochitosanase was purified and characterized from the culture supernatant of Streptomyces zaomyceticus C6, isolated from garden soil. It was a constitutive chitosanase producer, producing chitosanase in the culture medium even without chitosan as an inducer. The chitosanase was further purified to homogeneity by acetone fractionation followed by column chromatography. The molecular weight of chitosanase was found to be 95 kDa, using SDS-PAGE and gel filtration chromatography. The maximum velocity of chitosanase was attained at 60°C when the pH was maintained at 5.5. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.5, and about 20% of the initial activity was retained even after heating at 90°C for 10 min. The isoelectric point of the enzyme was found to be 6.5. Chitosanase showed wide substrate specificity towards chitosan substrates, but was not able to degrade chitin. An enzyme membrane reactor equipped with an ultra filtration membrane having a cut-off of 3000 Da was used for the fractionation of chitosan oligosaccharides produced by the purified chitosanase enzyme and an improved yield of chitosan pentamer (0.1 mg/ml) and hexamer (0.6 mg/ml) was achieved.

A facile and rapid method for green synthesis of Achyranthes aspera stem extract-mediated silver nano-composites with cidal potential against Aedes aegypti L.

Aedes aegypti L. is the primary vector associated with transmission of globally concerned diseases; Zika, yellow fever, dengue and Chikungunya. Present study investigates an efficient, alternative and comparative approach for mosquito control which is safe to environment and non-target organisms. The silver nano-composites (AgNCs) were synthesized from the aqueous stem extract of Achyranthes aspera (AASE) using different concentration of aqueous silver nitrate (AgNO3). The synthesis was tracked by UV-vis spectrophotometer and particle size analyser (DLS). The evaluation of their larvicidal potential against early fourth instars of Ae. aegypti showed significant potency, the toxicity increasing with the concentration of silver nitrate. The 24, 48 and 72 h bioassays resulted in respective LC50 values of 26.693, 1.113 and 0.610 μg/mL (3 mM AASE-AgNO3) 9.119, 0.420 and 0.407 μg/mL (4 mM AASE-AgNO3) and that of 4.283, 0.3 and 0.248 μg/mL (5 mM AASE-AgNO3). Keeping in view the significantly high larvicidal efficiency at lower concentration of silver nitrate, the 4 mM nano-composites were selected over 5 mM composites for further biophysical characterization carried out by X-ray Diffraction (XRD), Fourier transform infrared spectrometer (FTIR), Scanning electron microscopy (SEM), Energy dispersive X-ray (EDX) spectroscopy and Transmission electron microscopy (TEM). SEM and TEM confirmed the synthesis of spherical poly-dispersed AgNCs with average size ranging from 1–30 nm. Characterization through XRD showed the crystalline face-centered-cubic (fcc) structure of AgNCs with the highest intense peak obtained at 2θ value of 31.82°. FT-IR data suggests complex nature of AgNCs showing clearly defined peaks in different ranges. The present investigations recommend AgNCs of A. aspera stems as a low-cost and eco-friendly alternative to chemical insecticides for mosquito control.

Effects of Achyranthes aspera Extracts on the Survival and Midgut Histo-architecture of Aedes aegypti L. Early IV Instars

Received: May 3, 2018 Revised: August 13, 2018 Accepted: August 28, 2018 Abstract: Background: Aedes aegypti L.; one of the most important insect vectors in the world; transmits several diseases of concern; Zika, yellow fever, Chikungunya, dengue and dengue haemorrhagic fever. Despite multifarious problems on humans, non-targets and environment; caused by synthetic chemical insecticides; these are still the prime and preferred control measures against dengue vector. Alternative control strategies using eco-friendly and bio-degradable plant products are being explored.