ABSTRACT
Hydrophobins are a class of small cysteine rich surface active proteins produced exclusively by filamentous fungi. It forms a nano layer in the cell-water interface, thereby protecting the emerging fungal hyphae from surrounding water. Even though hydrophobins have similar functions in fungi, they share less sequence similarity. In the current study, we made a comparative study of the hydrophobin produced by the mushroom Pleurotus floridanus (PfH). Mushroom P. floridanus was cultured in PD broth. The hydrophobin was purified by foam fractionation and characterized in terms of molecular weight, solubility and glycosylation. In the RP-HPLC analysis, the hydrophobin eluted at a retention time of 45.56 min. The molecular weight of the PfH was found to be 13.52 kDa by MALDI-TOF MS and the LC-MS/MS showed no similar sequence in MASCOT database. The hydrophobin gene of P. floridanus was amplified using custom-designed primers and the BLAST analysis showed 80% sequence similarity with the Vmh2-1 gene of Pleurotus ostreatus. The sequence was translated into protein using ExPASy, secondary and tertiary structure predictions were carried out using Jpred4 and Phyre2. The tertiary structure showed 91.5% similarity with the HYD1 hydrophobin of Schizophyllum commune. A comparative study of PfH with Vmh2-1 and HYD1 was performed using bioinformatics tools. Hydrophobic cluster analysis revealed that three of these proteins have uniformity in terms of amphiphilic and non-amphiphilic α-helices, whereas PfH has a unique proline clustering. Physicochemical analysis by ProtParam revealed that PfH shares similar properties with HYD1 and Vmh2-1, which can be correlated with its function.
Subject(s)
Fungal Proteins , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Pleurotus/chemistry , Pleurotus/genetics , Pleurotus/metabolismABSTRACT
OBJECTIVES: An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina. RESULTS: BTPI-301was purified to homogeneity by (NH4)2SO4, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5 × 10-10 M. It was stable and active at pH 4-12 and also at 4-90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin. CONCLUSION: A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Protease Inhibitors/isolation & purification , Protease Inhibitors/metabolism , Pseudomonas mendocina/enzymology , Bacterial Proteins/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Protease Inhibitors/chemistry , Protein Stability , Trypsin/metabolism , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/isolation & purification , Trypsin Inhibitors/metabolismABSTRACT
Chitosan capped zinc sulphide nanocrystals doped with manganese (ZnS:Mn) have been synthesised by chemical capping co-precipitation method and structurally characterised by XRD, TEM and EDXS techniques. The dependence of optical properties on the size of these bio-compatible ZnS:Mn nanocrystals was investigated by UV/Vis and photoluminescence (PL) spectroscopic techniques in aqueous solvents. A variation in molar concentration of the precursor, sodium sulphide, from 0.125 to 0.01 mol L(-1) is accompanied by a decrease in particle size. The excitonic peak in the UV/Vis spectra is found to be blue shifted with a decrease in size of the nanocrystals due to confinement effects. In the present study, trypsin was immobilised onto ZnS:Mn nanocrystals using glutaraldehyde (GA) as cross-linker, which was confirmed by photoluminescence (PL) and Fourier transform infrared (FTIR) spectroscopic studies. Results indicate that the activity of trypsin, immobilised onto chitosan modified nanocrystals, has improved upon cross-linking, which suggests that the immobilised trypsin has become more stable and active. This work highlights the prospects of potential applications of immobilised trypsin in therapeutic and diagnostic fields.
