Molecular and Functional Characterization of a Novel Alcohol Oxidase From Aspergillus Terreus MTCC6324

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A novel flavin based alcohol oxidase (AOx) has been successfully cloned, sequenced and characterized from a hydrocarbon degrading filamentous fungi Aspergillus terreus MTCC 6324.The cDNA encoding AOx was constructed from mRNA of n-hexadecane induced cells of A.terreus MTCC6324 and expressed successfully as recombinant AOx (rAOx) in Escherichia coli. A combined proteomics and genomics approach was successfully performed to characterize an AOx coding open reading frame (ORF). Proteomics approach starts with the isolation of microsomal protein extract following a well established differential centrifugation protocol. Thereafter, the microsomal protein extract was resolved through 2D electrophoresis and the resolved 2D spots were manually processed for MALDI MS based peptide mass fingerprinting (pmf). Successful identification of target protein (AOx) through pmf database search was achieved. Two internal PCR primers were designed based on the wild type AOx internal peptide sequence match from pmf database search. Two internal PCR primers in combination with two PCR primers designed corresponding to the 5’ and 3’ end of hypothetical nucleotide sequence information of AOx from A.terreus NIH2624 in NCBI, proved successful in picking up two overlapping PCR amplicons flanking the full length coding region of AOx from A.terreus MTCC6324. The two overlapping PCR products were ligated at a common restriction endonuclease site present in its overlapping region to attain the full length 2001 bp ORF of AOx confirmed through double stranded primer walking which corresponds to 666 amino acid residues and the same had been submitted to NCBI Genbank with an accession no JX139751. The nucleotide and deduced amino acid sequence showed high sequence homology with unreviewed hypothetical AOx from A. terreus NIH2624 strain and maximum structural homology with chain B of aryl alcohol oxidase (AAO) from Pleurotus eryngii (PDB id: 3FIM). The full length coding region was successfully sub-cloned into E.coli expression vector and over-expressed as a recombinant histidine-tagged-AOx protein (rAOx). Due to the lack of proper post-translational modification in prokaryotic expression system, the over-expressed rAOx was found to be associated as highly aggregated, inclusion bodies present in the pellet fraction of the bacterial cell lysate. Inclusion body isolation, purification and solubilization had been successfully demonstrated by minor modification of an existing protocol from the literature. Approximately, 10 mg protein per 2.4 g wet cell weight per liter of induced culture was achieved after one step purification of Histidine-tagged-rAOx using Ni2+ affinity chromatography. Western blot of the purified fraction with anti-histidine antibody confirmed the expression of rAOx as a histidine tagged fusion protein. In-vitro re-folding to a functionally active AOx holoenzyme with its co-factor FAD was achieved after incubation of apo-rAOx (~ 10μg ml-1) for ~ 80 h at 16 ºC in refolding buffer (20 mM Tris-HCl buffer, pH 9.0, 2.5 mM glutathione oxidized, 1mM dithiothreitol, 35 % glycerol, 0.6 M urea and 0.08 mM FAD). Successful incorporation of FAD into the protein matrix was confirmed through fluorescence spectroscopy. The apoenzyme pI as predicted by 2D-electrophoresis and Zeta potential was 6.5±0.1 and mass as determined by MALDI-TOF/TOF was ~74 kDa, which was in good co-relation with the theoretical studies. Circular dichroism (CD) data of the refolded rAOx confirmed its ordered structure with α-helix, β-strand and random/non-regular structures as 28 ± 1%, 33 ± 2 % and 39 ± 2 %, respectively as predicted through CD spectra analysis program. The high aggregating nature of the rAOx had been demonstrated through dynamic light scattering studies and a significant shift in peak of the hydrodynamic radius to ~1000 nm within 24 h incubation in refolding condition was evident in our observation. Docking studies with an ab-initio protein model as predicted though online protein structure prediction software I-TASSER, demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The modeled rAOx was found to be specific for aryl alcohols and the order of its substrate preference with its total binding energy in parenthesis was evaluated as follows: 4-methoxybenzyl alcohol (-82.87 kJ mol-1) > 3-methoxybenzyl alcohol (-74.12 kJ mol-1)> 3, 4 dimethoxybenzyl alcohol (-71.75 kJ mol-1)> benzyl alcohol (-56.45 kJ mol-1). Thermal and pH stability of rAOx were studied. A range of alcohol substrates for activity of the rAOx was studied and found that only the aryl alcohol substrates showed detectable activity for the recombinant enzyme. Enzyme kinetics pertaining to its specific activity, Km, Kcat were evaluated which validated in-silico docking simulations. Thermo-inactivation studies pertaining to activation and deactivation energies were studied based on Arrhenius type equation. Enthalpy (ΔH), free energy (ΔG) and entropy (ΔS) of bioactive rAOx were investigated and reported in this thesis. This thesis highlights the molecular characterization of a full length coding nucleotide sequence of AOx from filamentous fungi following a combined proteomics and genomics approach. The significant sequence variation of the characterized A.terreus rAOx protein from the prevailing AOxs demonstrated the novelty of the protein although functionally it is closer to the reported aryl alcohol oxidase from a lignin degrading filamentous fungi. The work demonstrated in this thesis would advance the knowledge on the molecular characteristics of AOxs from filamentous fungi and will open the scope for the production of this industrially useful enzyme through recombinant DNA technology.
Supervisor: Pranab Goswami