Kinetic characterization, thermo-stability and Reactive Red 195A dye detoxifying properties of manganese peroxidase-coupled gelatin hydrogel.

An indigenous and industrially important manganese peroxidase (MnP) was isolated from solid-state bio-processing of wheat bran by white-rot fungal strain Ganoderma lucidum IBL-05 under pre-optimized growth conditions. Crude MnP extract was partially purified (2.34-fold) to apparent homogeneity by ammonium sulphate precipitation and dialysis. The homogeneous enzyme preparation was encapsulated on gelatin matrix using glutaraldehyde as a cross-linking agent. Optimal conditions for highest immobilization (82.5%) were: gelatin 20% (w/v), glutaraldehyde 0.25% (v/v) and 2 h activation time using 0.6 mg/mL of protein concentration. Gelatin-encapsulated MnP presented its maximum activity at pH 6.0 and 60 °C. Thermo-stability was considerably improved after immobilization. The optimally active MnP fraction was tested against MnSO4 as a substrate to calculate kinetic parameters. More than 90% decolorization of Sandal-fix Red C4BLN (Reactive Red 195A) dye was achieved with immobilized MnP in 5 h. It also preserved more than 50% of its original activity after the sixth reusability cycle. The water quality parameters (pH, chemical oxygen demand, total organic carbon) and cytotoxicity (brine shrimp and Daphnia magna) studies revealed the non-toxic nature of the bio-treated dye sample. A lower Km, higher Vmax, greater acidic and thermal-resistant up to 60 °C were the improved catalytic features of immobilized MnP suggesting its suitability for a variety of biotechnological applications.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Preparation of Poly[lactic-co-glycolic] Acid Nanospheres and Its Role in Hepatoma Cells.

Poly[lactic-co-glycolic] acid (PLGA) targeting nanoparticles AFP/PLGA/Dt386, loaded with Dt386 plasmid of diphtheria toxin gene, modified by Alpha fetoprotein (AFP) monoclonal antibody, is prepared. Its physical and chemical properties and its effect on HepG2 cells are studied. Firstly, Dt386 expression plasmid pET11a/Dt386 is constructed and PLGA nanoparticles are prepared by emulsion solvent evaporation (ESE). Scanning electron microscope (SEM) is used to observe its morphology. Laser Particle Sizer is used to measure the particle size. In addition, the encapsulation efficiency, drug loading and in vitro release rate of PLGA nanoparticles are measured. Carboxy fluorescein and rhodamine fluorescein are used to double label IgG/PLGA/Dt386 and AFP/PLGA/Dt386 nanospheres, respectively, the entry of nanospheres into HepG2 cells are observed at 3 h and 12 h. The effect of AFP/PLGA/Dt386 nanospheres on the migration of HepG2 cells is examined by wounding healing assay. Transwell chamber experiment is used to detect the effect of AFP/PLGA/Dt386 nanospheres on the invasion of HepG2 cells. MTT method is utilized to determine the inhibitory activity of nanoparticles on HepG2 cell proliferation. After treated with IgG/PLGA/Dt386 and AFP/PLGA/Dt386 nanoparticles for 48 hours, flow cytometry is used to detect the apoptosis rate and cell cycle of HepG2 cells in each group. The results show that the prepared nanospheres have regular morphology, flat surface, average particle size of 265.72±12.46 nm, zeta potential of -18.15 mV. The average entrapment efficiency and drug loading are 78.48±1.71% and 3.16±0.35%, respectively. The nanoparticles release slowly and stably in vitro. At the 10th day, the release rate reaches 75.13%. PLGA nanospheres can effectively protect DNA from nuclease degradation. The results show that AFP/PLGA/Dt386 nanospheres have biological targeting effect and can be enriched in cells. AFP/PLGA/Dt386 nanoparticles can significantly inhibit the migration, invasion and proliferation of HepG2 cells, and promote apoptosis.

Horseradish peroxidase immobilization by copolymerization into cross-linked polyacrylamide gel and its dye degradation and detoxification potential.

Owing to the ever-increasing environmental and health impacts associated with the discharge of dye-based effluents, effective remediation of industrial waste have become a top priority for the industrialists and environmental fraternity, around the world. Plant-based peroxidases represent a suitable bio-remediating agent for the effective treatment of original dyes or dye-based colored wastewater effluents. In the present study, horseradish peroxidase was immobilized by copolymerization into cross-linked polyacrylamide gel and investigated its potential for the degradation and detoxification of an azo dye, methyl orange. In the presence of N, N'-methylenebisacrylamide as a cross-linker, polyacrylamide gel at 10% concentration furnished >80% of immobilization yield. The surface morphology of the control (free enzyme) and immobilized enzyme, i.e., horseradish peroxidase cross-linked polyacrylamide gel was characterized using scanning electron microscopy. The high yielded horseradish peroxidase cross-linked polyacrylamide gel concentration, i.e., 10% was used to develop a packed bed reactor and exploited for dye degradation and detoxification purposes. A noteworthy level (>90%) of dye degradation was recorded after polyacrylamide gel cross-linked horseradish peroxidase-catalyzed reaction in a packed bed bioreactor. The biodegradation was further assessed by Fourier-transform infrared spectral analysis. The acute toxicity assays demonstrated that enzyme-based bio-degradation might be used effectively for large-scale environmental remediation of dyes and dyes containing wastewater effluents.

Biotransformation of lignocellulosic materials into value-added products-A review.

In the past decade, with the key biotechnological advancements, lignocellulosic materials have gained a particular importance. In serious consideration of global economic, environmental and energy issues, research scientists have been re-directing their interests in (re)-valorizing naturally occurring lignocellulosic-based materials. In this context, lignin-modifying enzymes (LMEs) have gained considerable attention in numerous industrial and biotechnological processes. However, their lower catalytic efficiencies and operational stabilities limit their practical and multipurpose applications in various sectors. Therefore, to expand the range of natural industrial biocatalysts e.g. LMEs, significant progress related to the enzyme biotechnology has appeared. Owing to the abundant lignocellulose availability along with LMEs in combination with the scientific advances in the biotechnological era, solid-phase biocatalysts can be economically tailored on a large scale. This review article outlines first briefly on the lignocellulose materials as a potential source for biotransformation into value-added products including composites, fine chemicals, nutraceutical, delignification, and enzymes. Comprehensive information is also given on the purification and characterization of LMEs to present their potential for the industrial and biotechnological sector.

Novel characteristics of horseradish peroxidase immobilized onto the polyvinyl alcohol-alginate beads and its methyl orange degradation potential.

Herein, we report the immobilization of in-house isolated horseradish peroxidase (HRP) from Armoracia rusticana with novel characteristics. The HRP was immobilized onto the self-fabricated polyvinyl alcohol-alginate (PVA-alginate) beads using sodium nitrate as a cross-linker. The PVA-alginate beads (2.0mm size) developed using 10% PVA and 1.5% sodium alginate showed maximal immobilization yield. The surface morphologies of the PVA-alginate (control) and immobilized-HRP were characterized by scanning electron microscopy (SEM). The immobilized-HRP retained 64.14% of its initial activity after 10 consecutive substrate-oxidation cycles as compared to the free counterpart. Simultaneously, the thermal stability of the immobilized-HRP was significantly enhanced as compared to the free HRP. The enzyme leakage (EL) assay was performed by storing the immobilized-HRP in phosphate buffer solution for 30days. Evidently, the leakage of immobilized-HRP was recorded to be 6.98% and 14.82% after 15 and 30days of incubation, respectively. Finally, the immobilized-HRP was used for methyl orange (MO) dye degradation in a batch mode. A noticeable decline in spectral shift accompanied by no appearance of a new peak demonstrated the complete degradation of MO. The degraded fragments of MO were scrutinized by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A plausible degradation pathway for MO was proposed based on the identified intermediates. In conclusion, the study portrays the PVA-alginate-immobilized-HRP as a cost-effective and industrially desirable green catalyst, for biotechnological at large and industrial in particular, especially for the treatment of textile dyes or dye-containing industrial waste effluents.