Micropollutants (ciprofloxacin and norfloxacin) remediation from wastewater through laccase derived from spent mushroom waste: Fate, toxicity, and degradation.

Fluoroquinolone antibiotics frequently found in environmental matrices (wastewater treatment plants, hospital wastewater, industrial wastewater and surface wastewater) causes potential threat to the environment. Enzymatic treatment for degradation of antibiotics from environmental matrices is a green and sustainable approach. Focusing on this, this study aimed to degrade two frequently found fluroquinolone emergent pollutants, ciprofloxacin and norfloxacin from wastewater. The trinuclear cluster of copper ions present in laccase has the ability to effectively remove organic micropollutants (OMPs). The uniqueness of this study is that it utilizes laccase enzyme extracted from spent mushroom waste (SMW) of P. florida for degradation of ciprofloxacin and norfloxacin and to achieve highest degradation efficiency various parameters were tweaked such as pH (3-6), temperature (30 °C and 50 °C), and ABTS (0.05, 0.6, and 1 mM) concentration. The results showed that the most effective degradation of ciprofloxacin (86.12-75.94%) and norfloxacin (83.27-65.94%) was achieved in 3 h at pH 4.5, temperature 30 °C, and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 0.05 mM concentration. Nevertheless, achieving degradation at 50 °C for both antibiotics, indicates thermostability nature of laccase (P. florida). Further, the fate of transformed products obtained from laccase mediated degradation was confirmed by liquid chromatography (LC-MS). Both the antibiotics undergo decarboxylation, depiperylyzation, dealkylation and defluorination as a result of laccase-mediated bond breakage. Anti-microbial activity of the biodegraded products was monitored by residual anti-bacterial toxicity test (E. coli and Staphylococcus aureus). The biodegraded products were found to be non-toxic and resulted in the growth of E. coli and Staphylococcus aureus, as determined by the agar-diffusion method. Moreover, the storage stability of laccase was determined for 28-day duration at varying pH (3-10) and temperature (4-50 °C). The maximum storage stability was obtained at pH 4.5 and temperature 30 °C. Therefore, utilizing SMW for the degradation of OMPs from wastewater not only benefits in degradation but also reuses SMW agro waste, shedding light on agro waste management. Thus, SMW is a one-pot solution for both OMPs biodegradation and circularity in the economy.

Label-Free Tracking of Hepatitis B Virus Core Protein Capsid Assembly in Real-Time Using Protein Charge Transfer Spectra.

Hepatitis B virions are double-shelled particles, with a diameter of 40-42 nm, consisting of a nucleocapsid called the HBV core protein (HBV Cp). It is an ordered assembly of 90-120 homodimers arranged in an icosahedral symmetry. Both the full-length HBV Cp and the first-149 residue domain, HBV Cp149, can spontaneously assemble in vitro into capsids with 120 Cp dimers (T = 4) or 90 Cp dimers (T = 3), triggered by high ionic strength of 0.25-0.5 M NaCl. The assembly disassembly of HBV Cp149 capsids are generally studied by light scattering, size-exclusion chromatography, atomic force microscopy, transmission electron microscopy, and other high-end expensive techniques. Here, we report a simple, yet robust, label-free technique exploiting protein charge transfer spectra (ProCharTS) to monitor the capsid assembly in real-time. ProCharTS absorption in the near UV-visible region (250-800 nm) arises when photoinduced electron transfer occurs from HOMO of COO- in glutamate (donor) to LUMO of NH3+ in lysine or polypeptide backbone (acceptor) of the protein. Alternatively, it can also occur from polypeptide backbone (donor) to acceptor in arginine, histidine, or lysine cation. ProCharTS is observed profusely among proximal charge clusters in folded proteins. Here, we show that, ProCharTS absorption among growing HBV capsids is amplified when HBV Cp homodimers assemble, generating new contacts among charged residues in the dimer-dimer interface. We notice a time-dependent sigmoidal increase in ProCharTS absorbance and luminescence during capsid formation in comparison to pure dimers. Additionally, a combined approach of anisotropy-based fluorescence assay is reported, where an increased fluorescence anisotropy was observed in capsids as compared to native and unfolded dimers. We conclude that ProCharTS can serve as a sensitive label-free tool for rapid tracking of capsid assembly in real-time and characterize the assembled capsids from dimers.

Optimization of laccase enzyme extraction from spent mushroom waste of Pleurotus florida through ANN-PSO modeling: An ecofriendly and economical approach.

The cardinal focus of this study is to optimize the best reaction conditions for maximizing laccase activity from spent mushroom waste (SMW) of Pleurotus florida. Optimization process parameters were studied by the modeling techniques, artificial neural networking (ANN) embedded in particle swarm optimization (PSO), and response surface model (RSM). The best topology of ANN-PSO architecture was obtained on 4-10-1. The R2, IOA, MSE, and MAE values of the ANN model were obtained as 0.98785, 0.9939, 0.0023, and 0.0251 while, that of the RSM model were obtained as 0.74290, 0.9210, 0.0244, and 0.1110 respectively. The higher values of R2, IOA, and lower values of MSE and MAE of the ANN-PSO model depict that ANN-PSO outperformed compared to RSM and also verified the effectiveness of the ANN-PSO model. The ANN-PSO model performance demonstrates the robustness of the technique in optimizing laccase activity in SMW of P. florida. The optimization results revealed that pH 4.5, time 3 h, solid: solution ratio 1:5, and ABTS concentration of 1 mM was optimal for achieving maximum laccase activity at temperature 30 °C. The enzymatic activity of crude laccase enzyme was obtained as 1.185 U ml-1 without loss of enzyme activity. Additionally, crude laccase enzyme was 1.74 fold partially purified, and 83.54% of the enzyme was yielded. Out of all the independent process variables, ABTS and pH had an influence on laccase activity. Therefore, we anticipate that the findings of this investigation will reduce the ambiguity in maximizing laccase activity and ease the screening process. This study also highlights the comparative cost evaluation of crude laccase enzyme extracted from P. florida and commercial enzymes. There is a great potential for the utilization of the laccase enzyme extracted from SMW and using it for the degradation of recalcitrant micropollutants. Thus, SMW promises a cost-effective and sustainable approach leading towards circular economy.