Purification, , characterization characterization Purification - - PowerPoint PPT Presentation

Ondrej Kaplan, Vojtech Vejvoda, Maria Cantarella, Agata Spera, Laura Cantarella, Ludmila Martinkova

Purification Purification, , characterization characterization and applications of and applications of a novel a novel nitrilase nitrilase from from Aspergillus niger Aspergillus niger

Department of Chemistry, Chemical Engineering and Materials, University of L`Aquila, L`Aquila, Italy Laboratory of Biotransformation, Institute

• f Microbiology, Academy of Sciences of the

Czech Republic

Laboratory of Biotransformation, Prague Screening of cultures, enzyme purification

Nitrilases

– well explored in bacteria bacteria – easy manipulation

• fast growth
• high activity
• broad substrate specifity

Occurence

• f nitrile-converting enzymes

Bacteria: Corynebacterium, Rhodococcus, Arthrobacter, Alcaligenes, Nocardia, Streptomyces, Pseudomonas Yeasts: Candida, Cryptococcus, Rhodotorula Filamentous fungi: Fusarium, Penicillium, Aspergillus, Myrothecium Plants: Brassica, Gramineae, Musaceae

Nitrilases

(nitrile aminohydrolases EC 3.5.5.X)

• mostly homooligomers
• do not involve any metal cofactor

3 types:

• aliphatic
• aromatic
• arylacetonitrilases

Our aim: To examine the biocatalytic potential

• f nitrilases from filamentous fungi

Aspergillus niger

• strain A. niger K10 was selected from screening of our

collection of filamentous fungi

• the culture grows in pellets

Hyperinduction of nitrilase Aspergillus niger K10

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Activity (U/mg of protein) . acetonitrile propionitrile butyronitrile isobutyronitrile valeronitrile 2-cyanopyridine 3-cyanopyridine 4-cyanopyridine

Step Proteins [mg] Specific activity [U/mg] Total activity [U] Yield [% ] Purification [fold]

Crude extract 114 1.7 190 100 1 Precipitation (NH4)2SO4 (40-50%) 15 8 127 67 5 Concentration/desalting 12 10 123 65 6 Q-Sepharose 1.1 114 122 64 69 Superdex 200 0.2 78 15.4 8.1 47

Activity assayed with 25 mM benzonitrile at 45°C and pH 8

Purification protocol

Nitrilase purification: SDS-PAGE electrophoreogram

1 2 3 4 5 6 7 8

1, 8… MARKER 2…… BSA STANDARD 3…… CRUDE EXTRACT 4…… AFTER PRECIPITATION 5…… AFTER CONCENTRATION / DESALTING 6…… AFTER Q-SEPHAROSE 7…… AFTER SUPERDEX 200

Enzyme subunit – 35 kDa

Optima of the nitrilase

20 40 60 80 100 5 7 9 11 13

pH Activity (%) .

20 40 60 80 100 15 25 35 45 55

Temperature (°C) Activity (%) .

Thermal

• ptimum; activity assayed

with 25 mM benzonitrile (pH 8.0, 50 mM Tris/HCl) pH Optimum; activity assayed with 25 mM benzonitrile (45°C, 100 mM Britton-Robinson buffer pH 5-13)

Substrate specifity of the nitrilase

Compound

Relative activity (% )

BENZONITRILE 100 INDOLYL-3-ACETONITRILE 2-HYDROXYBENZONITRILE 3-HYDROXYBENZONITRILE 2 4-HYDROXYBENZONITRILE 2-CHLOROBENZONITRILE 3-CHLOROBENZONITRILE 40 4-CHLOROBENZONITRILE 33 2-PHENYLACETONITRILE 9 2-PHENYLPROPIONITRILE 1 2-PHENYLBUTYRONITRILE 1

Compound

Relative activity (% )

2-TOLUNITRILE 3-TOLUNITRILE 7 4-TOLUNITRILE 1 2-CYANOPYRIDINE 30 3-CYANOPYRIDINE 48 4-CYANOPYRIDINE 355 ACETONITRILE PROPIONITRILE BUTYRONITRILE ISOBUTYRONITRILE VALERONITRILE

50 100 150 200 250 300

Specific activity (%) . controle 1 controle 2 BSA (1mg/mL) BSA (5mg/mL) Leupeptine (1mg/L) Glycerol (30%) Sucrose (30%) Mannitol (20%) Sorbitol (20%) Xylitol (20%) Trehalose (20%) Ethylene glycole (20%) EDTA (15mM) Sodium butyrate (25mM) Benzamide (25mM) Polyethylene glycole (1%) Polyvinyl alcohol (<1%) Urea (1%)

Controle 1 = initial activity, controle 2 = activity after 190 minutes at 45°C

Nitrilase stabilisers

10 20 30 40 50 60 70 80 90 100

Specific activity (%) .

• -- (Blank)

Methanol Ethanol 2-Propanol Acetone Acetonitrile Toluene DMSO Ethylacetate

Organic solvent conc. 5% v/v

Effect of cosolvents on nitrilase activity

Aspergillus niger K10

Experiments in a continuous ultrafiltration membrane reactor „University of L‘Aquila“, Italy

University of L‘Aquila

Continuous U-F membrane reactor

Cutting of the membrane for U-F membrane reactor

Continuous U-F membrane reactor

Continuous U-F membrane reactor

Continuous U-F membrane reactor, running experiment

The effect of temperature on biotransformation of benzonitrile to benzoic acid using of A. niger K10 nitrilase.

Substrate: 20 mM benzonitrile, pH 8 (50mM Tris/HCl pufr), 5% methanol, 1mM EDTA

0.00 0.02 0.04 0.06 0.08 10 20 30 40 50 60 70 80

t (h)

Benzoic acid ( mol/L) .

T=19°C T=35°C T=30°C T=40°C

Continuous U-F membrane reactor

The effect

• f

(NH4)2SO4

• n

biotransformation

• f

benzonitrile using of A. niger K10 nitrilase.

35°C, substrate: 20 mM benzonitrile, pH 8 (50mM Tris/HCl pufr), 5% methanol, 1mM EDTA; (amount of enzyme: 0,9 U per reactor run )

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 10 20 30 40 50 60 70

Hours Activity (10 -3 . U/mg of proteins).

No (NH4)2SO4 50mM (NH4)2SO4 100mM (NH4)2SO4 200mM (NH4)2SO4

Continuous U-F membrane reactor

Nitrilase stability

Nitrilase stored at different temperatures - 3-CP induced cells, enzyme purified by (NH4)2SO4 precipitation (40-65%)

0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 5 10 15 20 25

Days Activity (U/mg of protein).

• 69°C
• 18°C

+7°C +20°C

Thank you for your attention

Financial support:

National projects: A4020213 (Acad. Sci. Czech Rep.) OCD25.001 (Ministry of Education, Czech Rep.) ESF: Cost D25/0002/02