Amore, Antonella (2013) DEVELOPMENT OF NEW BIOCATALYSTS TO IMPROVE PRODUCTION OF SECOND GENERATION BIOETHANOL. [Tesi di dottorato]

[img]
Anteprima
Testo
Antonella_Amore_25.pdf

Download (25MB) | Anteprima
[error in script] [error in script]
Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: DEVELOPMENT OF NEW BIOCATALYSTS TO IMPROVE PRODUCTION OF SECOND GENERATION BIOETHANOL
Autori:
AutoreEmail
Amore, Antonellaa.amore@unina.it
Data: 30 Marzo 2013
Numero di pagine: 190
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Chimica organica e biochimica
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 25
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Faraco, Vincenzavfaraco@unina.it
Data: 30 Marzo 2013
Numero di pagine: 190
Parole chiave: Bioethanol; lignocellulose; cellulase; arabinofuranosidase; biorefinery
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 03 - Scienze chimiche > CHIM/11 - Chimica e biotecnologia delle fermentazioni
Aree tematiche (7° programma Quadro): BIOTECNOLOGIE, PRODOTTI ALIMENTARI E AGRICOLTURA > Produzione sostenibile e gestione delle risorse biologiche della terra, della foresta e dell'ambiente acquatico
ENERGIA > Produzione di combustibile rinnovabile
Depositato il: 03 Apr 2013 14:30
Ultima modifica: 10 Nov 2014 11:56
URI: http://www.fedoa.unina.it/id/eprint/9277

Abstract

Cellulases, hemicellulases (xylanases) and accessory enzymes (e.g.arabinofuranosidases, pectinases, mannanases) are needed for lignocellulose conversion into fermentable sugars for second generation bioethanol production. The high production costs of the above enzymes limit the bioethanol competitiveness,pushing towards the development of new biocatalysts, with improved catalytic properties, and new processes, whith lower costs. In this work, industrial waste based compost and natural habitats were investigated as a source of new (hemi)cellulolytic bacteria and fungi for discovery of new (hemi)cellulases. Tomato processing waste was shown a suitable substrate for the production of a new alpha-L-arabinofuranosidase (PoAbf) by solid state fermentation (SSF) with the basidiomycete Pleurotus ostreatus. Cellulolyitc activities of Bacillus amyloliquefaciens B31C and Streptomyces sp. G12 isolated from industrial waste based compost were characterized. An endoglucanase (CelB31C) from B.amyloliquefaciens B31C was identified, purified and characterized. The enzyme follows a Michaelis-Menten kinetics towards carboxymethyl-cellulose. It shows a retention of 90% of its activity for at least 6 days of incubation at 40 °C and a range of optimal temperatures from 50 to 70 °C. Investigating the enzymes responsible for cellulase activity produced by Streptomyces sp. G12, proteomic analyses led to the identification of three enzymes acting on cellulose. Gene coding for one of them, named CelStrep, was cloned and sequenced. Recombinant expression of CelStrep was carried out in Escherichia coli and the recombinant enzyme (rCelStrep) was characterized. rCelStrep follows a Michaelis-Menten kinetics towards carboxymethyl-cellulose and exhibits a half life of 24 h and 96 h at 60°C and 50°C, respectively and a retention of around 80% of activity after 96 h at 40°C. 93 microorganisms were isolated from 50 samples collected in the Western Ghat region (India, Kerala). Their screening for cellulases and xylanases production led to the selection of 7 xylanolytic and 14 cellulolytic microorganisms, identified by 16S rRNA sequencing as belonging to the species Bacillus, Streptomyces, Lysinobacillus and Paenibacillus. A further screening in liquid medium revealed the strain Lysinobacillus xylanolyticus XR84 able to produce both cellulase and xylanase activities. Due to the interest in thermo-resistant cellulases and to the ability of filamentous fungi to grow on agro-industrial wastes by SSF, 150 thermophilic filamentous fungi isolated from a "maasra" were screened for cellulase activity production at 45°C, leading to the selection of four Aspergillus strains, a Malbranchea strain and a Myceliophthora thermophila strain, all representing a potential reservoir of thermostable enzymes to be further identified and characterized. Proteomic analyses led to the identification of an alpha-L-arabinofuranosidase (PoAbf) responsible for the xylanase activity produced by P.ostreatus SSF on tomato waste. cDNA coding for PoAbf was synthesized and sequenced. Recombinant expression systems were set up in Kluyveromyces lactis and Pichia pastoris, the latter being the best host for enzyme production (180 mg L-1). Recombinant PoAbf (rPoAbf) follows Michaelis-Menten kinetics. It has a durable activity in a broad range of pH, particularly at pH 5 (t1/2= 51 days), which is also its optimal pH. It shows high stability (t1/2= 7 days) at 30°C and 40°C, the latter being its optimal temperature. It was shown to be a versatile enzyme, able to hydrolyze arabinooligosaccharides, arabinan and arabinoxylan. Glycosylation in position S160 positively affects the enzyme stability as demonstrated by site-directed mutagenesis experiments.

Actions (login required)

Modifica documento Modifica documento