oxygenase activities for the biosynthesis of aromatic antioxidants

124 Pages·2014·4.1 MB·English

OOXXYYGGEENNAASSEE AACCTTIIVVIITTIIEESS
FFOORR TTHHEE BBIIOOSSYYNNTTHHEESSIISS OOFF
AARROOMMAATTIICC AANNTTIIOOXXIIDDAANNTTSS

Giuliana Donadio

Dottorato in Scienze Biotecnologiche – XXVI° ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

Dottorato in Scienze Biotecnologiche – XXVI° ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

OOXXYYGGEENNAASSEE AACCTTIIVVIITTIIEESS
FFOORR TTHHEE BBIIOOSSYYNNTTHHEESSIISS
OOFF AARROOMMAATTIICC AANNTTIIOOXXIIDDAANNTTSS

Giuliana Donadio

Dottorando: Giuliana Donadio

Relatore: Prof. Alberto Di Donato

Coordinatore: Prof. Giovanni Sannia

"Vorrei tanto vedere un tramonto.. Ordinate al sole di
tramontare..." disse il Piccolo principe.
Rispose il re: "Se ordinassi a un generale di volare da un fiore all'altro
come una farfalla, o di scrivere una tragedia, o di trasformarsi in un
uccello marino; e se il generale non eseguisse l'ordine ricevuto, chi
avrebbe torto, lui o io?"
"L'avreste voi", disse con fermezza il piccolo principe.
"Esatto. Bisogna esigere da ciascuno quello che ciascuno puo' dare",
continuo' il re.
"L'autorita' riposa, prima di tutto, sulla ragione….

INDEX

Abstract PAG 5

Riassunto 7

Chapter 1 - Introduction 13
1.1 Oxidative stress in biological systems 14

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1.2 Phenols and catechols as antioxidants

1.3 Microbial biotransformations for the production of phenolic antioxidants 21

1.4 Bacterial Multicomponent Monooxygenases: ToMO as a case study 22

1.5 Baeyer-Villiger monooxygenases as biocatalysts 27

1.6 Bacillus subtilis in industrial biotransformations 30

1.7 Aim of the PhD project 32

Chapter 2 - Materials and methods 35

2.1 Bacterial strains 36

2.2 Bacterial culture media 36

2.3 Antibiotics 36
2.4 Proteins 36

2.5 Plasmids 36

2.6 Plasmidic DNA Purification 36
2.7 Purification of DNA fragments from agarose gel 36

2.8 Preparation of competent cells and transformation of E. coli 37

2.9 Preparation of competent cells and transformation of Bacillus subtilis 37

2.10 Genomic DNA extraction from Bacillus subtilis 37

2.11 Restriction endonucleases 37

2.12 Isolation of plasmid DNA from Bacillus subtilis 37

2.13 SDS-PAGE. 37
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2.14 Whole-cell assays 37

2.15 Catalytic assay on crude extract for identification of ToMO activity 38

2.16 Total RNA purification and RT-PCR 38

2.17 Identification of the reaction products of ToMO-catalyzed hydroxylation
of non-natural aromatic substrates 39

2.18 Purification of reaction products 39

2.19 NMR and mass spectrometry analysis of catechols 39

2.20 DPPH assay 40

2.21 Cell cultures and extracts 40

2.22 Immunofluorescence 40

2.23 MTT Assay 41

2.24 Ethidium bromide and acridine orange assay 41

Chapter 3 - Results and discussion Section I 43

3.1.1 ToMO from Pseudomonas sp. OX1 for the biosynthesis of phenolic 44
antioxidants

3.1.2. Identification and characterization of the products obtained from the 44
ToMO-catalyzed hydroxylation

3.1.3 In vitro assessment of the antioxidant potential: the DPPH assay 51

3.1.4 Cytotoxicity studies on H9c2 cell line 55

Chapter 3 – Results and discussion Section II 61

3.2.1 The Industrial scale-up of ToMO-catalyzed hydroxylation: use of 62
Bacillus subtilis as a GRAS host for the heterologous expression of
oxygenase activities

3.2.2 Cloning the tou gene cluster: integration into the genome of Bacillus 63
subtilis

3.2.3 Cloning of tou gene cluster: the use of the non-integrative plasmid 68
pHT01

2
3.2.4 Total RNA purification and RT-PCR 77

Chapter 3 – Results and discussion Section III 79
3.3.1. 3,6 diketocamphane 1,6-monooxygenase, a type II Baeyer-Villiger 80
Monooxygenases from Pseudomonas putida NCIMB 10007

3.3.2. Expression and purification of 3,6-diketocamphane 1,6 82
monooxygenase (3,6 DKCMO)

3.3.3 Crystallization of 3,6-diketocamphane 1,6 monooxygenase (3,6 85
DKCMO)

3.3.4 ThermoFluor Assay on 3,6-DKCMO 88

3.3.5 Cloning, expression and purification of the flavin reductase 90
component of 3,6-DKCMO

Conclusions 95

References 99

Appendices 105

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