Thermomyces lanuginosus: A prospective thermophilic fungus for green synthesis and stabilization of BioAgNPs through glucoamylase

Hypothesis: The rise of bionanoscience has led to the use of silver nanoparticles in therapeutic and industrial applications like biomedicine, biocatalysis, bioremediation and biosensing. Enhancement of metal nanoparticle stability is of critical significance when it comes to safe handling and efficient use of nanoparticles for various applications. In this regard, thermophilic fungi is a potential candidate with their massive thermostable excretome titres. This work aims to explore the potential of thermophilic fungi to provide surface stability to biogenic silver nanoparticles (BioAgNPs). Experiment: Thermophilic fungus Thermomyces lanuginosus STm was used to synthesize BioAgNPs. The size and shape of BioAgNPs were characterized using ultraviolet-visible spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction techniques. The most significant process variables affecting mycosynthesis of BioAgNPs were screened using statistical experimental design of Placket Berman model. The mechanism of mycosynthesis and capping on biogenic nanoparticles was explored using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-mass spectrometry (LC-MS/MS). Findings: Characterization of BioAgNPs by ultraviolet-visible spectroscopy revealed surface plasmon resonance at 410 nm, X-ray diffraction calculated 7-24 nm diameter of BioAgNPs and transmission electron microscopy showed BioAgNPs to be spherical with 5-35 nm size range. The physico-chemical factors affecting synthesis of BioAgNPs were screened by statistical modeling using Placket Berman experiment design which revealed the significant role of pH and silver precursor salt concentration on BioAgNPs synthesis. The supramolecular bound protein moiety was initially screened using FTIR analysis. The protein coating around BioAgNPs was found to be 67 kDa glucoamylase when detached from NP and investigated using SDS-PAGE and LCMS/MS analysis. In vitro cytotoxic assay against eukaryotic animal model Artemia salina (brine shrimp) larvae revealed dose dependent decrease in viability indicating BioAgNPs to be less cytotoxic, as compared to silver in ionic form. Conclusion: Glucoamylase protein (67 kDa) secreted extracellularly by T. lanuginosus STm was discovered to be the capping protein surrounding BioAgNPs. It is interesting to note that in case of BioAgNPs synthesized by extracellular titre of thermophilic fungus, the adsorbed thermostable protein glucoamylase can possibly help in retaining activity of BioAgNPs at elevated temperature as per the industrial requirement. Thus it can be a natural way of immobilization of enzyme on the surface of nanoparticles while eliminating the extra steps require for immobilizing for commercial uses. In terms of acute toxicity, BioAgNPs were found to be less toxic and safer as compared to ionic silver.

Automated summary

The research discovered that the 67 kDa glucoamylase secreted by thermophilic fungus T. lanuginosus STm can act as a capping protein for biogenic silver nanoparticles (BioAgNPs), enhancing their thermal stability and biological activity, making them suitable for industrial applications. In addition, BioAgNPs were found to be less toxic compared to ionic silver.