Production, purification, and characterization of p-diphenol oxidase (PDO) enzyme from lignolytic fungal isolate Schizophyllum commune MF-O5.

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.

Diversity in Elemental Content in Selected Artemisia L. (Asteraceae) Species from Gilgit-Baltistan Region of Pakistan Based on Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES).

Diversity in eleven Artemisia species from northern Pakistan was assessed based on as per suitability of their elemental contents with thermal conductivity detection and ICP-AES procedures. Results indicated the presence of 13 major elements in the Artemisia species with varied concentrations including Carbon (45.7%, 45,7000 ppm-49.8%, 49,8000 ppm), Nitrogen (2.03%, 20,300 ppm-3.50%, 35,000 ppm), Phosphorus (0.168%, 1680 ppm-0.642%, 6420 ppm), Potassium (2.38%, 23,800 ppm-4.72%, 47,200 ppm), Sulphur (1920 ppm, 0.192%-4780 ppm, 0.478%), Boron (23.8 ppm, 0.00238%-71.7 ppm, 0.00717%), Calcium (0.733%, 7330 ppm-2.249%, 22,490 ppm), Magnesium (0.116%, 1160 ppm-0.267%, 2670 ppm), Zinc (27.7 ppm, 0.00277%-47.9 ppm, 0.00479%), Manganese (25.7 ppm, 0.00257%-93.8 ppm, 0.00938%), Iron (353 ppm, 0.0353%-1532 ppm, 0.1532%), Copper (14.1 ppm, 0.00141%-26.2 ppm, 0.00262%) and Sodium (105 ppm, 0.0105%-587 ppm, 0.0587%). Cluster analysis distributed the Artemisia species into two major groups (G1 and G2) on the basis of their elemental content where G1 contained species like, Artemisia herba alba Asso., A. tournefortiana Rachb., A. rutifolia Steph. ex Spreng., and A. vulgaris L., with the presence of all elements with the maximum amount of S, Zn, P, Ca, and Mg, while G2 contained species like Artemisia biennis Willd., A. chamaemelifolia Vill., A. capillaris, L., A. gmelinii Weber ex Stech., A. indica Willd., A. maritima L., and A. verlotiorum Lamotte., with all elements but significant concentrations of B, N, C, K, Mn, Fe, Cu, and Na. PCA analysis displayed maximum species diversity in the axes two, while axes one showed lower diversity. Additionally, the elevated levels of elements recorded as compared to the threshold levels recommended in the literature for medicinal plants require extraordinary precautionary measures before or during using Artemisia as medication to avoid metal toxicity.