Isolation, purification, characterization and antimicrobial activity of antimicrobial peptides isolated from Lentinus squarrosulus cultivated in Thailand

Abstract

The emergence of microbial pathogenic resistance to current antibiotic drugs has increased the interest in developing alternative antibiotics from natural resources. Antimicrobial peptides (AMPs), a family of proteins from various organisms with antimicrobial activity, may be potential new antibiotic drugs which can be utilized to overcome the antibiotic resistance. The aim of this study is to isolate, purify and characterize the AMP from Lentinus squarrosulus (Mont.), the common edible mushroom cultivated in Thailand. Crude proteins were extracted from fresh fruiting body by homogenization with 10 mM Tris-HCl buffer, pH 7.4 and the proteins were precipitated by solid ammonium sulfate at the final concentration of 40-80% (w/v). The crude protein extracts were further purified by anion exchange chromatography using DEAE - cellulose column. The partial purified protein collecting from anion exchange column using the elution buffer (0.5 M NaCl in 10 mM Tris-HCl buffer, pH 7.4) showed the antifungal activity. After further purification by Sephadex G-25 gel filtration, the active protein was eluted in the fourth peak. The antimicrobial activity test using agar disc diffusion method exhibited the strong activity against various species of human fungal pathogen including Candida albicans ATCC 10231, Candida tropicalis, Trichophyton mentagrophytes and Trichophyton Rubrum which the last 3 species were clinical isolates. However, the activity against Aspergillus niger was low. Furthermore, it had no activity against both gram positive and gram negative bacteria as well as no synergistic effect with antibiotic drugs (ciprofloxacin, chloramphenicol and tetracycline). The antifungal activity of protein was completely lost in the presence of proteases, the pH above or below the range of 3.0 - 9.0 and the temperature above 40 oC. The nuclease activity was also detected on both genomic DNA and plasmid DNA. The purified protein had the molecular mass of approximately 17 kilodalton determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis under reducing condition. Amino acid sequence determined by MALDI-TOF/MS showed high similarity to the conserved sequences of cyclophilin, the protein with the antifungal property. The findings from this study suggested that antifungal protein/peptide from L. squarrosulus (Mont.) may have potential clinical application which need further investigation.