Appendix

Table A1. Culture medium for bacteria

Media Constituents Concentration (g l^-1) pH Luria Bertani broth

(LB)

Casein enzymic hydrolysate Yeast extract

NaCl

10.0

5.0 10.0

7.5±0.2

Luria Bertani agar (LB)

Casein enzymic hydrolysate Yeast extract

NaCl Agar

10.0

5.0 10.0 15.0

7.5±0.2

Note: Both the above bacterial media were procured as a readymade powdered media from HIMEDIA Laboratories (India). LB broth and LB agar were prepared by mixing 25.0 g l^-1 and 40.0 g l^-1, respectively and autoclaved at 15 lb (121⁰C) for 15 min before inoculating with bacterial culture.

Table A2. Composition of n-hexadecane basal medium

Components Concentration (g l^-1)

MgSO4.7H2O 0.2

NH4NO3 1.0

CaCl2 0.02

KH2PO4 1.0

K2HPO4 1.0

FeCl3 0.002

Yeast extract 1.0

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Note: The components were mixed and the pH of the medium was adjusted to 5.8. 50 ml of basal medium was transferred to 500 ml Erlenmeyer flasks and 2% (v/v) n-hexadecane was added to the medium for AOx induction. The media containing the substrate was sterilized at 15 lb (121⁰C) for 15 min before inoculating with fungal mycelium.

Table A3. Composition of A.terreus lysis buffer

Components Concentration

EDTA 10 mM

MgCl2 1 mM

PMSF 1 mM

Note: The above components were added to 50 mM Tris/HCl buffer, pH 8.0 and after disrupting the cells, the cell homogenate was immediately mixed with 1 mM DTT. Do not autoclave the lysis buffer.

Table A4. List of buffers

Buffers/solutions Concentration Composition pH

Tris/HCl buffer 50 mM Tris base (6.057 g l^-1) 8.0 with 1N HCl MOPS electrophoresis

buffer

10X stock MOPS (200 mM, pH 7.0) Sodium acetate (80 mM)

EDTA (10 mM, pH 8.0)

8.0

TAE-Tris Acetate EDTA buffer

50X , 100 ml stock

Tris base (24.2 g) Glacial acetic acid (5.71 ml) 0.5M EDTA, pH 8.0 (10 ml)

8.0

Tris-glycine SDS gel running buffer

10X, 1 l stock Tris base (30.0 g) Glycine (144.0 g)

SDS (10.0 g)

8.3

Sodium phosphate buffer 100 ml, 0.2 M (stock)

19 ml 0.2 M NaH₂PO₄ (solution A) 81 ml 0.2 M Na2HPO4 (solution B)

7.4 Binding buffer for Ni²⁺

affinity chromatography

NA 20 mM sodium phosphate buffer pH 7.4, 500 mM NaCl, 4 M urea and 20

mM immidazole

7.4

Elution buffer for Ni²⁺

affinity chromatography

NA 20 mM sodium phosphate buffer pH 7.4, 500 mM NaCl, 4M urea and 500

mM immidazole

7.4

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Note: Binding buffer and elution buffer used for Ni2+ affinity chromatography were filter sterilized with 0.22 or 0.45 micron filter. NA stands for not applicable.

Table A5. Transformation and storage solution for bacterial competent cell preparation

Components Concentration

LB broth (pH 6.1) Appropriate volume as required

PEG (MW ~ 3350) 10 % (w/v)

MgCl₂ 10 mM

MgSO₄ 10 mM

Note: Adjust the pH of the final solution to pH 6.5, autoclave at 15 lb (121⁰C) for 15 min and store at 4⁰C.

Table A6. List of oligonucleotide PCR primers

Sl No Name Sequence (5’- 3’)

1 GAPDH-F CAAGGTCATCCATGACAACTTTG

2 GAPDH-R GTCCACCACCCTGTTGCTGTAG

3 AOX-FP1 ATGACTATTCCAGACGAAGTCGACA

4 AOX-RP1 TGAGACCAGGATCCATGCTGGAT

5 AOX-FP2 ACAGTCCCCTCCAAGCCGCT

6 AOX-RP2 TTACAGTCGAGCAAGCCCAGTAAACTC

7 AOX-pET28a-F GCCGAATTCATGACTATTCCAGACGAAG

8 AOX-pET28a-R CGCAAGCTTCAGTCGAGCAAGCCCAGT

Note: Restriction sites added to the PCR primers are highlighted in bold with its base pairs underlined.

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Table A7. Solutions for plasmid isolation

Solutions Composition pH

Solution I 50 mM glucose, 25 mM Tris/HCl buffer (pH 8.0), 10

mM EDTA (pH 8.0)

8.0

Solution II NaOH (freshly diluted from a 10N stock), 1% (w/v) SDS

___

Solution III 60.0 ml Potassium acetate (5M stock), 11.5 ml glacial acetic acid, 28.5 ml ddH2O

___

Note: Solution I should be prepared in batch of ~100 ml, autoclaved at 15 lb (121⁰C) for 15 min and store at 4⁰C. Solution II should be prepared fresh and use at room temperature. Solution III should not be autoclaved and stored at 4⁰C. Solution I and III should be chilled on ice prior to use.

Table A8. List of restriction enzymes

Restriction

Enzyme

Recognition site

100%

activity in NEB buffer

Temperature of incubation

(⁰C)

Time of incubation

Heat inactivation

(⁰C)

Heat inactivation

time

EcoRI-HF (high fidelity)

G/AATTC Buffer 4

37 5 h 65 20 min

Bg1II A/GATCT Buffer 3

37 5 h No __

NdeI CA/TATG Buffer 4

37 5 h 65 20 min

HindIII-HF (high fidelity)

A/AGCTT Buffer 4

37 5 h 80 20 min

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Table A9. Composition of bacterial lysis buffer

Components Concentration

Sodium phosphate buffer (pH 7.4) 20 mM

DTT 5 mM

PMSF 1 mM

Note: DTT and PMSF were added just prior to use and no autoclave is required.

Table A10. List of antibiotics

Name Stock Concentration

(mg ml^-1)

Solvent Working

concentration (µg ml^-1)

Ampicillin 100 ddH2O 50-100

Kanamycin 50 ddH2O 25-50

Note: All the antibiotic was filter sterilized and appropriate aliquots were stored at -20⁰C until further use. Repeated freeze/thaw cycles should be avoided.

Table A11. Buffer/solutions for SDS-PAGE

Buffers/solutions Compositions

30 % acrylamide-bisacrylamide solution (100 ml)

29.2 g acrylamide, 0.8 g bisacrylamide 0.5 M Tris/HCl pH 6.8 (100 ml) 6.06 g of Tris base, pH adjusted to 6.8 with

2 N HCl.

1.5 M Tris/HCl pH 8.8 (100 ml) 18.18 g Tris base, pH adjusted to 8.8 with 2 N HCl.

Tris-glycine SDS gel running buffer Appendix Table A4

SDS gel loading buffer (2X) 100 mM Tris/HCl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) Bromophenol blue, 20% (v/v) glycerol, 200 mM DTT or β-mercaptoethanol Note: DTT if used should be added just prior to use.

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Table A12. Blue silver staining solution for SDS-PAGE

Components Concentration

DdH2O Required amount

Ortho phosphoric acid 10 % (v/v)

Ammonium sulfate 10 % (w/v)

CBB G250 0.12% (w/v)

Methanol 20% (v/v)

Note: Add the following components in the order as given in the table. An initial volume of water was taken to dissolve the components and later the volume was make-up to the final desired volume by adding more ddH2O.

Table A13. Buffers /solutions used for Western Blot

Buffers/solutions Components

Protein transfer buffer 25 mM Tris base, 39 mM glycine, 20% (v/v) methanol

Phosphate buffer saline (PBS) (1 l), pH7.5 11.5 g Di-sodium hydrogen orthophosphate, 2.96 g Sodium dihydrogen orthophosphate,

5.84 g Sodium chloride Phosphate buffer saline with Tween 20,

washing buffer (PBS-T)

Add 0.05% (v/v) Tween 20 to PBS

Blocking solution 3% (w/v) BSA in PBS-T

Table A14. List of antibodies

Name Source/Type Working

condition

Working dilution

Use Anti-poly-histidine

antibody

Mouse/monoclonal RT/2 h 1:3000 Primary

antibody in western blot Anti-mouse IgG

(Fab specific)- peroxidase antibody

Goat/monoclonal RT/1 h 1:6000 Secondary

antibody in western blot

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Table A15. List of scoring parameters in I-TASSER

Scoring function Detailed description

Confidence score (C-score) Confidence score for estimating the quality of predicted models by I-TASSER. Significance of threading template alignments and the convergence parameters of the structure assembly simulations C-score is typically in the range of [-5,2], where a C-score of higher value signifies a model with a high confidence and vice-versa

Template modeling score (TM-score) Scale for measuring the structural similarity between two structures. The small distance is weighted stronger than the big distance. A TM- score > 0.5 indicates a model of correct topology and a TM-score < 0.17 means a random similarity. The cut off independent of protein length.

Root mean square deviation (RMSD) Parameter for measuring structural similarity between two structures which are usually used to measure the accuracy of structure modeling when the native structure is known. Where the native structure is not known, distance between the predicted model and the native structure predicts the quality of the modeling prediction.

RMSD is an average distance of all residue pairs in two structures.

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Table A16. List of plasmid vectors

Name Company Use Promoter Selection

marker (s)

Cloning site

pGEM-T Easy

Promega T/A cloning vector

T7 lac promoter

Amp^r EcoRI site

pET-28a (+) Novagen Bacterial expression

vector

T7 lac promoter

Kan^r Multiple

cloning site (BamHI- XhoI)

Figure A1. Vector maps of plasmids used

Figure A1.A. Plasmid map of pGEM-T Easy T/A cloning vector (adapted from www.promega.com)

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Figure A1.B. Plasmid map of pET28a (+) bacterial expression vector (adapted from www.novagen.com)

Publications

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