Size exclusion chromatography for the removal of pigments from extracellular ligninolytic enzyme extracts from decayed wheat straw

Solid-state fermentation of wheat straw was carried out by a native white rot basidiomycete Daedaleopsis flavida strain 5A. Extract prepared from the 12-day decayed wheat straw contained extracellular ligninolytic enzymes like manganese peroxidase (MnP), manganese-independent peroxidase (MIP), lignin peroxidase (LiP) and laccase along with straw-degraded products and pigments. Sephacryl S-200 size exclusion chromatography in 16/100 column was used for the separation of these ligninolytic enzymes and straw-degraded products and pigments. Recovery of pigment-free ligninolytic enzyme activities as protein was 40% of the total proteins loaded and specific LiP activity increased 34 fold after size exclusion chromatography. Thus accurate estimation of LiP by veratryl alcohol oxidation assay was possible only after the removal of interfering pigments. The reproducibility of size exclusion chromatography is adjudged satisfactory from the consistent results obtained after seven repetitive uses of matrices.     

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.