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Purification, , characterization characterization Purification and applications of a novel a novel nitrilase nitrilase and applications of from Aspergillus niger Aspergillus niger from Ondrej Kaplan, Vojtech Vejvoda, Maria Cantarella, Agata Spera, Laura Cantarella, Ludmila Martinkova Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic Department of Chemistry, Chemical Engineering and Materials, University of L`Aquila, L`Aquila, Italy

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 of 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 of 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 Activity (U/mg of protein) . Aspergillus niger K10 0.0 0.5 1.0 1.5 2.0 2.5 3.0 acetonitrile propionitrile butyronitrile isobutyronitrile valeronitrile 2-cyanopyridine 3-cyanopyridine 4-cyanopyridine

Purification protocol Proteins Specific Total Yield Purification [mg] activity activity [% ] [fold] Step [U/mg] [U] Crude extract 114 1.7 190 100 1 Precipitation (NH 4 ) 2 SO 4 (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

Nitrilase purification: SDS-PAGE electrophoreogram Enzyme subunit – 35 kDa 1, 8… MARKER 2…… BSA STANDARD 3…… CRUDE EXTRACT 4…… AFTER PRECIPITATION 5…… AFTER CONCENTRATION / DESALTING 6…… AFTER Q-SEPHAROSE 7…… AFTER SUPERDEX 200 1 2 3 4 5 6 7 8

Optima of the nitrilase 100 pH Optimum; activity assayed with 25 mM benzonitrile (45°C, 100 mM 80 Activity (%) . Britton-Robinson buffer pH 5-13) 60 40 20 0 5 7 9 11 13 pH 100 80 Activity (%) . 60 40 Thermal optimum; activity assayed with 25 mM benzonitrile (pH 8.0, 20 50 mM Tris/HCl) 0 15 25 35 45 55 Temperature (°C)

Substrate specifity of the nitrilase Relative Relative Compound Compound activity (% ) activity (% ) BENZONITRILE 100 2-TOLUNITRILE 0 INDOLYL-3-ACETONITRILE 0 3-TOLUNITRILE 7 2-HYDROXYBENZONITRILE 0 4-TOLUNITRILE 1 3-HYDROXYBENZONITRILE 2 2-CYANOPYRIDINE 30 4-HYDROXYBENZONITRILE 0 3-CYANOPYRIDINE 48 2-CHLOROBENZONITRILE 0 4-CYANOPYRIDINE 355 3-CHLOROBENZONITRILE 40 ACETONITRILE 0 4-CHLOROBENZONITRILE 33 PROPIONITRILE 0 2-PHENYLACETONITRILE 9 BUTYRONITRILE 0 2-PHENYLPROPIONITRILE 1 ISOBUTYRONITRILE 0 2-PHENYLBUTYRONITRILE 1 VALERONITRILE 0

controle 2 = activity after 190 minutes at 45°C Controle 1 = initial activity, Specific activity (%) . 100 150 200 250 300 50 0 controle 1 controle 2 BSA (1mg/mL) Nitrilase stabilisers 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%)

Effect of cosolvents on nitrilase activity 100 90 80 Specific activity (%) . 70 60 50 40 30 20 10 0 Methanol Ethanol Ethylacetate --- (Blank) 2-Propanol Acetonitrile Toluene DMSO Acetone Organic solvent conc. 5% v/v

Aspergillus niger K10 „University of L‘Aquila“, Italy Experiments in a continuous ultrafiltration membrane reactor

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

Continuous U-F membrane reactor 0.08 0.06 mol/L) . 0.04 Benzoic acid ( 0.02 0.00 0 10 20 30 40 50 60 70 80 t (h) T=19°C T=35°C T=30°C T=40°C 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

Continuous U-F membrane reactor 0.80 Activity (10 -3 . U/mg of proteins). 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 10 20 30 40 50 60 70 Hours No (NH4)2SO4 50mM (NH4)2SO4 100mM (NH4)2SO4 200mM (NH4)2SO4 The effect of (NH 4 ) 2 SO 4 on biotransformation of 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 )

Nitrilase stability 0.100 0.090 0.080 Activity (U/mg of protein). 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 0 5 10 15 20 25 -69°C -18°C Days +7°C +20°C Nitrilase stored at different temperatures - 3-CP induced cells, enzyme purified by (NH 4 ) 2 SO 4 precipitation (40-65%)

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