The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

Three-dimensional microspheric g-C3N4 coupled by Broussonetia papyrifera biochar: facile sodium alginate immobilization and excellent photocatalytic Cr(iv) reduction

g-C3N4–Broussonetia papyrifera biochar–sodium alginate composites were prepared. Composite recyclability and activity for photocatalytic Cr(vi) reduction and the origin of enhanced photocatalytic activity relative to that of g-C3N4 were probed.

Immobilization and some application of α-amylase purified from Rhizoctonia solani AG-4 strain ZB-34

Background Aim of the study was to immobilize the α-amylase produced earlier from the mesophilic fungus Rhizoctonia solani AG-4 strain ZB-34 by solid-state fermentation and investigate the suitability of immobilized enzymes for some industries. Materials and methods A novel α-amylase from R. solani AG-4 strain ZB-34 was immobilized in chitosan by covalent binding and Ca-alginate by entrapment. Results The efficiency of chitosan and Ca-alginate immobilization was 67.9% and 59.6%, respectively. The immobilized enzymes showed the highest activity in the presence of starch. Optimum values for chitosan and Ca-alginate immobilized enzymes were pH 4.50 and 40°C and pH 5.50 and 60°C, respectively. It was found that immobilized enzymes were highly stable in terms of thermal and pH stabilities. When the chitosan immobilized enzyme was used with detergents, chocolate stains on dirty laundry was better cleaned. Chitosan immobilized R. solani AG-4 strain ZB-34 α-amylase was found to have a higher desizing effect at 40°C in tap water. As a result of Ca-alginate immobilization, the enzyme clarified apple juice more than the free enzyme. Conclusion The results showed that immobilized enzymes might have potential applications in industry. This is the first report immobilizing an α-amylase produced from the fungus R. solani.

Immobilized and Free Cells of Geotrichum candidum for Asymmetric Reduction of Ketones: Stability and Recyclability

Marine-derived fungus Geotrichum candidum AS 2.361 was previously reported by our group as an active strain for the enantioselective reduction of ketones. Although some other Geotrichum strains were also found from the terrestrial sources, information on their stability and reusability is scarce. Herein, the stabilities—in terms of pH tolerance, thermostability, and storage stability, and reusability—of G. candidum AS 2.361 were described for the asymmetric reduction of a series of aromatic ketones. Two differently immobilized cells (agar immobilization and calcium alginate immobilization) as well as free cells were prepared. For three substrates (1-(3-bromophenyl) ethan-1-one (1b), 1-(2-chlorophenyl) ethan-1-one (1d), and acetophenone (1g)) immobilized cells on agar showed a great improvement in the bioreduction activities compared to the free cells, increasing yields up to 97% with ee values of 99%. Cells immobilized on agar/calcium alginate could maintain more than 90% of the original activities within the assayed pH ranges of 3.5–11, while free cells were highly sensitive to alkaline and acidic conditions. Concerning thermostability, immobilized cells on agar kept 99% of their original activities after incubation at 60 °C for 1 h, while almost no activity was detected for the free cells under the same condition. Immobilized cells were stable at 4 °C for 80 days without any activity loss, while free cells started to decrease the activity after storage at 4 °C for six days. The immobilized cells retained almost 99% activity after four reuse cycles, while free cells lost almost all the activities at on the third cycle.

Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability

Composite materials based on a titanium support and a thin, alginate hydrogel could be used in bone tissue engineering as a scaffold material that provides biologically active molecules. The main objective of this contribution is to characterize the activation and the functionalization of titanium surfaces by the covalent immobilization of anchoring layers of self-assembled bisphosphonate neridronate monolayers and polymer films of 3-aminopropyltriethoxysilane and biomimetic poly(dopamine). These were further used to bind a bio-functional alginate coating. The success of the titanium surface activation, anchoring layer formation and alginate immobilization, as well as the stability upon immersion under physiological-like conditions, are demonstrated by different surface sensitive techniques such as spectroscopic ellipsometry, infrared reflection–absorption spectroscopy and X-ray photoelectron spectroscopy. The changes in morphology and the established continuity of the layers are examined by scanning electron microscopy, surface profilometry and atomic force microscopy. The changes in hydrophilicity after each modification step are further examined by contact angle goniometry.

Enhanced Production of Ethanol from Cheese whey by Agarose and Alginate immobilization of Yeast Cells

Yeasts cells isolated from dry yeast were immobilized in alginate and agarose to form beads. The fermentation of ethanol from cheese whey was optimized with respect to temperature, pH, and rpm. A comparative study was performed between immobilized and free cells to get the maximum ethanol production Results revealed that 35°C temperature, 4.5 pH and 60 rpm is the optimized condition for yeast immobilized in agarose bead. Similarly, 35°C temperature, 5.0 pH, 60 rpm is the optimized condition for yeast immobilized in alginate bead and 35°C temperature, 5.0 pH, 80 rpm is the optimized condition for the free yeast cell respectively. Immobilised cell resulted 4.25% ethanol with alginate bead whereas 4.35% with agarose bead and 4.30% with free cell after three days of incubation under optimized conditions. When the same cell immobilized beads were reused, obtained 1.8% ethanol with agarose and 1.7% ethanol with alginate bead.DOI: http://dx.doi.org/10.3126/jncs.v30i0.9388Journal of Nepal Chemical Society Vol. 30, 2012 Page: 159-164 Uploaded date: 12/20/2013