I am highly indebted to my lab members, Seraj, Madhuri, Somashekhar, Ankana, Santhosh, Babina, Priyamvada, Priyanki, Mrinal, Sharbani, Naveen and Danish for their kind cooperation and making my lab research memorable and entertaining. I am grateful to my co-supervisor students Kokila, Amit, Subhamoy, Amaresh, Nidhi, Chokalingam, Archita, Sharmila, Asif, Neha and Upashi, and for extending their helping hand during my research work and making me feel as part of their research group.
61 Chapter 4: Results and Discussions
List of Abbreviations and Symbols
4.19 (A) Multiple N-terminal sequence alignment of AOx-encoding amino acid sequences from 11 species of filamentous fungi and yeast species and (B) Phylogenetic tree of the corresponding AOxs by Neighbor. 4.24 (A) 3D overlay of the best pose of FAD predicted in our modeled AOx binding simulation and (B) 3D protein overlay of the FAD-bound crystal structure of aryl AOx (IDB id: 3FIM) with FAD .
List of Tables
Abstract
Western blotting of the purified fraction with anti-histidine antibody confirmed the expression of rAOx as a histidine-tagged fusion protein. A series of alcohol substrates for activity of the rAOx were studied and found that only the aryl alcohol substrates showed detectable activity for the recombinant enzyme.
Introduction
The various AOx activities were localized in the microsomal membrane of the cells, and all activities were restricted to highly aggregated protein particles purified from the cell homogenate by chromatographic methods. This confused protein conglomerate of the enzymes, their intriguing metabolic role in the cells of A.
Literature Review
- Alcohol oxidase: Discovery and detection methods
- Types of alcohol oxidases
- Long chain alcohol oxidase
- Aromatic alcohol oxidase
- Secondary alcohol oxidase
- Characteristics of Aspergillus terreus
- Current protocols to characterize novel genes in filamentous fungi
In this line, β-naphthylmethanol was identified as the most easily oxidized substrate (Ferreira et al., 2005). The other sources for the enzyme are limited, such as Penicillium sp (Qian et al., 2004), Sphingopyxis sp.
Materials and Experimental Methodologies 3.A Materials
3.B Experimental Methodologies
3.B.1.2 Isolation and optimization of total RNA from A. terreus MTCC6324 Thin, papery, translucent mycelium from 4-day-old static culture of A. In subsequent steps of total RNA isolation, the manufacturer's protocol for plant RNA isolation was optimized with TRI reagent.
3.B.1.3 Quantification of RNA
AOx activity was assayed spectrophotometrically using the horseradish peroxidase-based coupled assay method of Kemp et al., (1988) that measured H 2 O 2 generation. Microsomal protein extract was dissolved in 50 mM Tris/HCl buffer (pH 8.0), dialyzed against ice-cold 20 mM Tris/HCl buffer (pH 8.0) supplemented with 0.1 mM PMSF for 4 h, and was estimated by its total protein concentration by the standard Bradford assay (Bradford, 1976) using bovine serum albumin as a standard protein.
3.B.1.10 Two dimentional (2D) gel electrophoresis
Total protein concentration was estimated by taking the optical density (O.D) values at 595 nm in the Multilabel 2030 reader (PerkinElmer, USA).
3.B.1.11 Mass spectrometry compatible silver staining of SDS-PAGE gel
3.B.1.12 Target protein identification from 2D gel spots
Target Protein Identification by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (4800 plus MALDI TOF/TOF Analyzer, AB SCIEX, USA) was performed for each of the 2D gel spots (Fig 3.3 step 6). For each 2D sample point, the peak list was generated using the explorer 4000 series software package (AB SCIEX, USA).
3.B.1.13 Peptide mass fingerprinting database search
An experimental overview of steps involved in the identification of target proteins from 2D gel by MALDI-TOF-MS. The target protein match was confirmed by searching the database for pmf data corresponding to each 2D spot analyzed (Fig. 3.3, step 9).
Molecular characterization of AOx (fragment 2) pmf data corresponding to the conserved region of msa and a reverse primer (AOx-RP2) (Appendix Table A6, Fig. 6) designed from the 3'-end of the AOx sequence information of A. terreus NIH2624 available in the NCBI database.
3.B.1.16 Overlapping PCR
PCR amplifications for both overlapping PCR products were performed using the GeneAmp PCR 9700 system (Applied Biosystems, USA) following optimized initial denaturation conditions at 94 ºC for 5 min; 35 cycles of amplification [a denaturation step at 94 ºC for 30 sec, annealing at (60 ºC for PCR 1 and 63 °C for second nested PCR 2) for 45 s, initial extension at 72 ºC for 1.5 min]; final extension at 72ºC for 10 min.
3.B.1.17 Agarose gel electrophoresis of DNA
3.B.1.18 Elution of DNA samples from agarose gel
GenElute binding column was placed in one of the 2 ml sterile collection tubes provided with the kit and equilibrated with 500 µl of column preparation solution (supplied with the kit) and centrifuged for 1 minute at 14,000 x g. The DNA was eluted by adding 50 µl of elution solution (supplied with the kit) preheated to 65 °C placed in the middle of the binding column in a fresh sterile Eppendorf tube (supplied with the kit) and centrifuged at 14,000 x g for 1 min.
3.B.1.19 Cloning PCR amplicons in pGEM-T Easy T/A vector system
The sample DNA solution was then loaded onto the pre-equilibrated binding column in a maximum volume not exceeding 700 µl at a time and centrifuged for 1 min at 14,000 x g. A blank centrifuge of the binding column-collection tube assembly was performed at 14,000 x g for 1 min to remove excess ethanol without adding more wash solution to it.
3.B.1.20 Quantification of DNA
The reaction mixture was pipetted and allowed to incubate at 4 °C in a circulating water bath overnight, and the ligated mixture was transformed into competent E.coli DH5a cells by the standard heat shock method described in Section 3.B.1.22 (Green and Sambrook, 2012 ) and plated on LB-agar/ampicillin/IPTG/X-Gal plates.
3.B.1.21 Competent E.coli cells preparation
A 1 % primary culture was aseptically loaded into a 25 ml LB broth and grown to early exponential phase until the optical density (O.D) at 660 nm wavelength read 0.4-0.5. The bacterial pellet was then aseptically resuspended in 2.5 ml of sterile ice-cold transformation and storage solution (TSS solution) (Appendix Table A5) and aliquoted into an appropriate volume and immediately frozen at -80 °C for long-term storage.
3.B.1.22 Transformation of competent E.coli cells
The competent cells were then subjected to heat shock at 42 °C in a circulating water bath for 90 s and immediately cooled on ice. The resuspended cells were plated on LB agar/IPTG/X-Gal plates containing the appropriate antibiotic and incubated for 12–16 hours at 37°C.
3.B.1.23 Plasmid DNA isolation
800 µl of prewarmed sterile LB broth at ~37 °C was aseptically added to the cells and allowed to incubate for 1 h at 37 °C with constant shaking at ~180 rpm. RNA contamination was removed by incubating the plasmid preparation with 20 µg ml-1 DNase-free RNase at 37 °C for 30 minutes.
3.B.1.24 Digestion of DNA by restriction enzymes
The supernatant was gently removed and the DNA pellet was washed with 70% ice-cold molecular biology grade ethanol. The plasmid DNA was resuspended in appropriate amount of nuclease free water for downstream application.
The same was also confirmed by double-stranded primer walking of a full-length AOx fragment cloned into the T/A vector. Schematic representation of the experimental methodology to confirm the orientation of the individual PCR fragments cloned into the T/A vector and subsequent ligation at the common restriction site (Bg1II) to form a full-length AOx clone into the T/A vector to be obtained.
3.B.1.26 DNA sequencing of cloned fragments
Molecular characterization of AOx sequencing was performed by the DNA Sequencing Facility, Department of Biochemistry, University of Delhi, South Campus using a 3130xl Genetic Analyzer (Applied Biosystems, USA).
Subcloning and expression of rAOx eluted and purified for downstream experimentation using GenElute gel extraction kit (Sigma Aldrich, USA) according to the manufacturer's protocol described in section 3.B.1.18. Successful ligation into pET28a(+) and transformation into BL21 (DE3) was confirmed by fragment release after restriction digestion with EcoRI and HindIII restriction enzymes according to the protocol previously described in section 3.B.1.24.
Subcloning and expressing rAOx LB medium at a 1:100 dilution as secondary culture and growing under the same conditions as above until the optical density (O.D) at λ 660 nm reached the mean logarithm of the phase (O.D for induction with IPTG. To check the overexpression of the protein , of interest, 5 ml of IPTG-induced cultures were harvested by centrifugation at 5000 x g for 10 min at 4 °C in sterile 1.5 ml or 2 ml polypropylene tubes.
To optimize the small-scale expression of apo-rAOx, the transformed secondary culture (5 ml volume) was induced with IPTG at a concentration of mM and a time point of 0, 4 and 8 h duration. Isolation, digestion and purification of apo-rAOx 3.B.3 Isolation, digestion and purification of apo-rAOx from inclusion.
3.B.3.2 Purification of solubilized rAOx by nickel affinity chromatography
Isolation, digestion and purification of apo-rAOx eluted with elution buffer (Appendix Table A4) at a flow rate of 2 ml min-1. SDS-PAGE of the eluted fractions was performed according to the standard Laemmli protocol described in section 3.B.2.4 to qualitatively control the purification of our histidine-tagged protein (N-terminal-6xHis-AOx-6xHis-C-terminal ).
3.B.3.3 Western blot analysis
3.B.3.4 In-vitro Refolding and Refolding of apo-rAOx with FAD Cofactor The in-vitro activation and refolding of purified apo-rAOx with its FAD cofactor were the in-vitro activation and refolding of cofactor-purified apo-rAOx his FAD was performed according to the protocol of Ruiz-Dueñas et al, (2005) (Fig. 3.9). A schematic overview showing the in-vitro refolding of apo-rAOx with its cofactor FAD in refolding buffer.
3.B.4.1 MALDI-TOF/TOF mass spectrometry of apo-rAOX
The plate was aligned and calibrated with the TOF/TOF calibration mixture (Applied Biosystem, AB SCIEX, USA) prepared according to the manufacturer's instructions before our sample analysis (Fig. 3.10 step 4 and 5). The MS/MS database search was performed by the ProteinPilot program (AB SCIEX, USA) (Fig. 3.10 step 5 & 6).
3.B.4.3 Determining isoelectric point (pI) of rAOx by Zeta potential
3.B.4.2 Determination of isoelectric point (pI) of rAOx by 2-D electrophoresis The theoretical pI was calculated using the online Compute pI/Mw tool (ExPASY. Thus a point where the plot of zeta potential versus pH gradient passes through zero zeta potential would reconfirm the isoelectric point of the protein.
The characteristic decrease of refolded rAOx-FAD holoenzyme spectra compared to free FAD indicates efficient incorporation of FAD into the protein matrix.
3.B.4.5 Circular dichroism (CD) spectroscopy measurement
DLS) analysis of rAOx
A schematic overview of studying the aggregation pattern of rAOx as it refolds in the refolding buffer after a regular 24-h interval for 96 h.
3.B.5.1 In-silico sequence analysis of AOx from A.terreus MTCC6324
3.B.5.2 Ab-initio based rAOx protein structure prediction
Templates used by I-TASSER to predict the model were also analyzed for the stereochemical quality of all protein chains in a given PDB structure, as a control study.
3.B.6.1 Determining enzyme activity and kinetics
A schematic overview of determining rAOX activity and kinetic parameters by monitoring the change in absorption of the product over time.
Functional characterization of rAOx mol -1 K-1 was calculated from Arrhenius type equation (equation 1) using the following derived equations 2, 3 and 4 respectively. First-order reaction constant is the slope of the regression line obtained by plotting (ln V) against time at different temperatures expressed in kJ mol-1.
3.B.6.3 Measuring the half life and decimal reduction values of rAOx
The quality of cDNA prepared from each of these isolated total RNA samples was verified by amplification of the housekeeping gene GAPDH corresponding to the ~496 bp amplicon (Fig. 4.2). The quality of cDNA produced from total RNA samples isolated by the following protocol (b) was similar to that produced by the commercial kit, as judged by their band intensity on the gel.
4.A.1.2 Designing primers from the peptide sequences of AOx protein
Alcohol oxidase cDNA was constructed from total RNA isolated from n-hexadecane-induced A.terreus MTCC6324 cells following the protocol as mentioned in Experimental Methodology Section 3.B.1.2. 4.A.1.3 Construction of full-length AOx gene by overlapping PCR approach AOx cDNA constructed from total RNA isolated from n-hexadecane.
Note to Table 4.2: The protocols followed for the comparative study are (a) Extraction of total RNA by the modified CTAB-based method (Zeng and Yang, 2002). Agarose formaldehyde gel electrophoresis of total RNA isolated from A.terreus MTCC6324 following five isolation techniques.
4.A.2.2 Expression, purification and in-vitro activation of rAOx
After purification, in-vitro refolding of apo-rAOx with its cofactor FAD was performed. Refolding was performed using ~10 μg ml-1 of purified apo-rAOx in refolding buffer.
4.A.3 Characterizations of physicochemical properties of rAOx
4.A.3.1 Characterization of apo-rAOx mass
4.A.3.2 Isoelectric point (pI) of rAOx
4.A.3.4 Analysis of secondary structure of the rAOx
4.A.3.5 Studies on the aggregating nature of rAOX
Panel B.2 shows the constant cAOx accumulation profile with no major peak shift when compared to the 0 h data. Panel C.3 shows the aggregation profile of BSA at 48 h showing the constant non-aggregating nature of the protein.
4.A.4.1 In-silico sequence analysis of rAOx
The results validated the novelty of the rAOx protein sequence derived from the characterized rAOx gene. A successful 3D model of the characterized rAOx has been generated and the results regarding its stereochemical validation are shown via the Ramachandran plot, as described in subsection 4.A.4.2.
4.A.4.2 Molecular modeling and docking studies of rAOx
Amino acid sequence identity (using NCBI BLAST) of A. terreus AOx with other aryl AOxs from P. simplicissimum showed 25 % and 37 %, respectively, and weak homology was found in the N-terminal region of the protein. The residuals lying in the additional allowed, generously allowed and forbidden areas of the plot are 2.8% respectively.
4.A.5 Steady state kinetics and thermo-inactivation studies of rAOx
4.A.5.1 Enzyme activity and kinetic studies of AOx
Such high variation of activation energies indicates the conformational changes especially in the substrate binding site that improve the affinity of rAOx for substrate (aromatic alcohols) binding. The larger slope of line (a) and smaller slope of line (b) indicate high and low activation energies associated in the respective temperature ranges and thus the rate constants associated are high and less sensitively derived, respectively.
4.A.5.3 Thermoinactivation studies of rAOx
Elucidation of kinetic deactivation parameters at different incubation temperatures ranging from 30 °C to 70 °C of the purified rAOx. The slope of the plot of natural logarithm of kd values versus T-1 (plot area B) predicted the conformational deactivation energy (Ead.
4.B Discussions
The cDNA sequence of aryl AOx from A.terreus that we derived consists of 666 amino acids, while the widely studied aryl AOx from P. It also confirmed that the active site is in close proximity to the isoalloxazine ring of FAD.
Bibliography
Dickinson FM, Wadforth C (1992) Purification and some properties of alcohol oxidase from Candida tropicalis grown in alkane. Gvozdev AR, Tukhvatullin IA, Gvozdev RI (2010) Purification and properties of alcohol oxidase from Pichia putida.
Appendix
Root Mean Square Deviation (RMSD) A parameter to measure the structural similarity between two structures, commonly used to measure the accuracy of structure modeling when the source structure is known. If the source structure is unknown, the distance between the predicted model and the source structure predicts the quality of the modeling prediction.
List of Publications
In Referred Journals
NCBI/GenBank submission
Abstracts Published in Conferences
