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Mineralization of Synthetic Lignins in the Presence of a Phenol Oxidase Activity and Hydroxyl Radicals Produced by Petriellidium fusoideum. Petriellidium.

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Presentation on theme: "Mineralization of Synthetic Lignins in the Presence of a Phenol Oxidase Activity and Hydroxyl Radicals Produced by Petriellidium fusoideum. Petriellidium."— Presentation transcript:

1 Mineralization of Synthetic Lignins in the Presence of a Phenol Oxidase Activity and Hydroxyl Radicals Produced by Petriellidium fusoideum. Petriellidium fusoideum MUCL Isolation from Soil Samples Collected in Peruvian Amazonia 14 C-MWL-Wheat Straw 37% 14 CO 2 Optimal ligninolytic conditions: C/N=12, T=28ºC, pH=4,5 Dehydropolymerised Coniferyl Alcohol (DHP)

2 dpm Days mU / mL Laccase Activity Mineralization of a Synthetic Lignin with thioglycolate control

3 Detection of HO. radicals in fungal cultures 1)Enoki et al., (1989) Target substrate: 2-keto thiomethyl butyric acid (KTBA) Substrate produced: Ethylene 2)Babbs and Gale (1987) Target substrate: Dimethyl sulphoxide (DMSO) Substrate produced: Methyl sulphinic acid

4 Ethylene Generation and Mineralization of a Synthetic Lignin in P. fusoideum Cultures Days ppm dpm

5 Mineralization of a Synthetic Lignin in P. fusoideum Cultures Control After addition benzoate (1 mM) Days dpm

6 Low molecular weight substance containing cation metals. Single electron oxidation activity. Hirano (1995); Tanaka et al., (1996; 1999) ; Enoki et al., (1997) O 2 O 2. - H 2 O 2. OH LMW LMW substance containing reduced cation LMW substance containing oxidized cation Electron source

7 Day 2Day 3Day 4 Abs max x Kav x Abs x Area of LMW-S Detected by Gel Filtration (Sephadex G-50) in P. fusoideum Cultures

8 ppm dpm Laccase Activity, MWL-substance and Ethylene Production Compared with the Mineralization in P. fusoideum Cultures Days mU / mL Abs max x 10

9 Summary of the results The fungus studied produced: 1)A laccase activity as only enzyme related to lignin degradation 2). OH radical detected by ethylene and methyl sulphinic acid production MW = 1000 Da 3)LMW-SNo protein content Fe %

10 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL)

11 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL) ppm Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen 1 st assay 2 nd assay 3 rd assay 4 th assay 5 th assay 6 th assay Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL) 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL) 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL) 1 st System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Ascorbic acid (1 mM)  LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL)

12 2 nd System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Hydroquinone (500   LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL)

13 2 nd System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Hydroquinone (500   LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL)  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen ppm 1 st assay 2 nd assay 3 rd assay4 th assay5 th assay6 th assay  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen  Super oxide dismutase Scavengers:  Sodium benzoate (10 mM)  Thiourea (10 mM)  Catalase (6 U)  Nitrogen 2 nd System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Hydroquinone (500   LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL) 2 nd System:  KTBA (3 mM)  Acetate buffer pH 4.5  Oxygen purging  Hydroquinone (500   LMW-S (1 mg / mL)  P. fusoideum laccase (1.62 mU / mL)

14 H 2 O. OH H 2 O 2 O 2 O 2. - KTBA Ethylene H2OH2O O2O2 LMW-S Fe 3+ LMW-S Fe 2+ MYCELIUM Q.-Q.- Q 2- Laccase R Laccase Q Fe 3+ + O 2.- Fe 2+ + O 2 H 2 O 2 + Fe 2+ Fe 3+ + OH. + OH - H 2 O 2 + O 2.- O 2 + OH. + OH -


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