Università Degli Studi Di Napoli “Federico II”
ENERBIOCHEM PON01_01966
New biocatalysts for
lignocelluloses conversion
Vincenza Faraco
Associate researcher of Department of Chemical Sciences (DSC) “BMA group”
Assistant professor of Environmental Biotechnology and Fermentation Chemistry
School of Biotechnological Sciences
Università Degli Studi Di Napoli “Federico II” (UNINA)
E-mail address vfaraco@unina.it
phone + 39 081674315/679895
mobile phone +39 3403483751
fax +39 081674313
SIEE 2012, 20 November 2012 Naples
Research institution profile
Department of Chemical Sciences
The department belonging to the University of Napoli Federico II employs a total of 89 members
including full professors, associate professors, assistant professors and researchers.
Areas of basic scientific research are: synthetic chemistry, carbohydrate chemistry, natural
compounds structure elucidation, and mechanics of organic reactions; biochemistry, molecular
biology, and proteomics.
The department allows access to several research facilities: NMR Spectrometers, 2D
electrophoresis and image analysis system, HPLC systems, Molecular graphics computer
systems, Silicon Graphics, UV-VIS-NIR spectrophotometer, polarimeter; diffractometer; Mass
spectrometers, DSC, NMR, WAXS, SEC, FT-IR, (DRIFT); UV-VIS (DRUV); Sorptomatic(BET);
TPD-TPRO; GC FID-TCD; HPLC UV-RI-Light Scattering.
Monte S’Angelo
Research Outlines
•Catching new better (hemi)cellulolytic, ligninolytic and accessory
enzymes for lignocellulosic biomass conversion by both purification from
new microorganims and metagenomic approaches and ethanol, biopolymers
and chemicals production;
•Elucidating structure-function relationships of (hemi)cellulolytic,
ligninolytic and accessory enzymes by rational mutagenesis;
•Developing and characterizing new biocatalysts by rational and random
mutagenesis;
•Developing bio-systems based on microorganisms and their enzymes for
lignocellulosic biomass and waste conversion for production of fuel
ethanol, biopolymers and fine/bulk chemicals;
•Developing bio-systems based on microbes and their enzymes for soil
and wastewater bioremediation;
Funded Projects
•
Industrial Research Project “Integrated agro-industrial chains with
high energy efficiency for the development of eco-compatible
processes of energy and biochemicals production from renewable
sources and for the land valorisation-ENERBIOCHEM” PON01_01966
(2011-2014) funded by Ministry of University and Scientific Research in the
frame of National Operative Programme Research and Competitiveness
2007–2013;
UNINA (V. Faraco) is leader of WP3 (Development of processes with
high energetic efficiency for industrial transformation of lignocellulosic biomasses, including waste valorisation) and member of
executive board.
•
Research Project funded by European Union in the frame of Seventh
Framework Programme (Marie Curie Actions People International Research
Staff Exchange Scheme) 2012-2016 “Improvement of technologies and
tools, e.g. biosystems and biocatalysts, for waste conversion to
develop an assortment of high added value eco-friendly and costeffective bio-products”-BIOASSORT
UNINA (V. Faraco) is coordinator
Research unit profile-equipment
As far as Molecular Biology research activity is concerned, BMA laboratories are equipped with basic equipment
for DNA preparation, cloning and analyses, and with Termo-cyclers –Thermal Gradient Cycler PTC-200 Peltier,
Celbio; MyCycler Thermal Cycler, Bio-Rad- for Polymerase Chain Reactions.
As far as Biochemistry, Bioinformatics and Mass Spectrometry research activity are concerned, BMA group is
endowed with facilities for:
-protein purification in specialized laboratories containing automated chromatographic systems, such as SMART
Chromatographic system, AKTA FPLC Chromatographic system, AKTA Explorer 100 Chromatographic system,
BIOPILOT Chromatographic system.
-screening of enzyme collections by using multi-well plates reader micro-technology (Microplate
Spectrophotometer Benchmark Plus, Bio-Rad), and Biomek® 3000 Laboratory Automation Workstation (Beckman
Coulter).
-mass spectrometry analyses in specialized laboratories for bio-analytical mass spectrometry (MS), where
advanced methods and strategies, based on MS, for the detection and characterization of biological molecules,
are developed. BMA mass spectrometry facilities include: VOYAGER DE STR laser de-sorption ionization
(MALDI) mass spectrometer equipped with a TOF-REFLECTRON analyzer; WATERS MICROMASS ZQ 2000 LCMS system equipped with a Z-SPRAY source and a quadrupole analyzer coupled with a WATERS Millenium
HPLC apparatus for LC-MS/MS analysis; VOYAGER DE PRO laser de-sorption ionization (MALDI) mass
spectrometer equipped with a TOF-REFLECTRON analyzer; Hybrid Q-Trap 4000 LC-MS/MS system coupled with
a capillary HPLC and electro-spray source.
As far as Microbiology is concerned BMA laboratories are equipped with a 28 °C thermostatic room for fungi and
bacteria growth, incubators, orbital shakers, and laminar flow cabinets.
As far as Fermentation technology is concerned, BMA group is endowed with a specialized laboratory equipped
by a Multiple Automatically controlled bioreactor (4x500ml, Sixfors, Infors) that allows screening of microbial
fermentation conditions; Automatically controlled bioreactor for microbial fermentation up to 2 liters (Applicon);
Automatically controlled bioreactor for microbial fermentation up to 7 liters (Techfors S, Infors).
For Analyses of lignocellulosic biomasses, BMA group is endowed with the system FiberCap™ 2021/2023 (Foss)
for analyses of lignin, cellulose and hemicellulose contents, fibres and ash.
2nd Generation Bioethanol
It is produced from lignocellulose biomass, differently from 1st generation bioethanol
produced from edible agricultural materials easily fermentable
Advantages
 Lignocellulose represents the most abundant biomass, 1,5 trilion ton/year
 Lignocellulosic raw materials are geographically more evenly distributed
 Lignocellulosic raw materials minimize the potential food-versus-fuel conflict
 Using lignocellulose is less expensive than using conventional agricultural feedstock
 Biofuels from lignocellulose generate low net greenhouse gas emissions
Problems
 Structure complexity of biomass
 Highly costs of lignocellulose comversion
 High cost of lignocellulolytic enzymes
 Low energy yield
2nd Generation Bioethanol from lignocellulose
Lignocellulose
Highly complex structure, mainly composed of
cellulose, hemicellulose and lignin
Alcohol fermenting
microorganisms
Lignin to Energy
Pre-treatments
for lignin removal
(Hemi)cellulose
Hydrolysis
• Chemical/Physical
treatments
• Chemical hydrolysis
(Hemi)Cellulases are needed for an efficient
lignocellulosic biomass hydrolysis step.
• Enzymatic hydrolysis
Bio-ethanol
The major cost driving factor in
lignocellulosic biomass-to-ethanol process is
the cost of (hemi)cellulolytic enzymes.
Sustainable
Energy
C5/C6-Sugars
Fermentation
Main challenges..
…..to increase 2nd generation bioethanol production competitiveness
♦ REDUCTION OF COSTS ASSOCIATED TO ENZYMATIC HYDROLYSIS STEP
♦ Reducing cellulolytic/hemicellulolyitc enzymes costs of production
♦ Improving cellulolytic/hemicellulolyitc enzymes performances
(catalytic efficiency, extreme pH and high-temperature resistence/stability)
♦ INCREASE OF FERMENTABLE SUGARS YIELDS: HEMICELLULOSE HYDROLYSIS
♦ New hemicellulases with enhanced performances
♦ DEVELOPMENT OF A BIOMASS BIOREFINERY
«a facility that integrates biomass conversion processes and equipment to produce fuels,
power, and value-added chemicals from biomass » (NREL)
Project tasks
Identification and
characterization of
new
(hemi)cellulolytic,
ligninolytic,
pectinolytic
microorganisms
INCREASE OF
FERMENTABLE
SUGARS YIELD
DEVELOPMENT OF
BIOREFINERY
CONCEPT
Identification and
characterization of
new glycosyl
hydrolases,
ligninases ,
pectinases
TASK 3
Set up of lignocellulosic biomass
valorization processes by the new
biocatalysts/biosystems
TASK 2
TASK 1
REDUCTION OF COSTS
ASSOCIATED TO
ENZYMATIC HYDROLYSIS
STEP
TASKS 1-2
Identification and characterization of new
(hemi)cellulolytic microorganisms and enzymes
Strategy I: Cultivation-dependent approach
Available
collections of
microorganisms
belonging to DSA,
IRD-IMEP, NIIST
New microbial
collections from
numerous habitats
Endogenous
microflora isolated
from biomasses
Optimization of enzyme production in
liquid culture of the selected microorganisms
Isolation and
selection of new
microrganisms*
Identification of selected enzymatic
activities by proteomics**
Recombinant expression of selected
enzyme in an appropriate host
*by team of Prof.Olimpia Pepe of Department of Food Science (DSA) of University of Naples “Federico II”
** in collaboration with team of Prof. Leila Birolo of Department of Chemistry Science (DSC) of University of Naples “Federico II”
TASK 1
NEW CELLULOLYTIC FUNGI*
Screening of 150 thermophilic filamentous fungi for
cellulase activity production
Selection of 9 fungi
Aspergillus spp., Myceliophtora thermophila, Malbranchea sp.
Setting up of a preliminary solid state fermentation (SSF)
process
1
3
4
* In collaboration with Prof. S. Roussos IRDIMEP Institute, Université Paul Cézanne, Aix
Marseille III
Raimbault’s columns
2
Raimbault, M. and Alazard, D. Culture method to study fungal growth in solid fermentation.
(1980) Eur. J. Appl. Microbial. Biotechnol. 9:199-209
TASK 1
NEW CELLULOLYTIC BACTERIA*
Screening of 180 bacteria from raw composting material for
cellulase activity production and selection of 19 strains
Phenotypic characterization
(colony morphology, gram reaction, catalase/oxidase reaction)
Time course of AZO-CMCase activity in liquid medium
and selection of 8 microorganims
(0.03-0.11 U/mL of AZO-CMCase activity)
Microorganisms identificatiom
(DNA fingerprinting and 16S rRNA gene sequencing)
ENERBIOCHEM PON01_01966
in collaboration with Prof. Olimpia Pepe, University of Naples “Federico II”
TASK 2
Identification and characterization of new glycosyl hydrolases
Bacillus amyloliquefaciens sp. B31C
Streptomyces sp. G12
 Zymogram analyses
Streptomyces sp. G12 Bacillus sp. B31C
 Proteomics analyses*
Protein
Endoglucanase from Bacillus
amyloliquefaciens
Cellulase from Streptomyces
xylophagus
Endoglucanase from
Streptomyces halstedii
Purification and characterization of the
endoglucanase from Bacillus
amyloliquefaciens sp. B31C
Coning and recombinant expression of
the gene codifying for Streptomyces sp.
G12 glycosyl hydrolase cellstrep in
Escherichia coli, characterization of the
recombinant enzyme.
Cellulose binding protein from
Streptomyces griseoflavus
ENERBIOCHEM PON01_01966
* in collaboration with Prof. Leila Birolo, DSC, University of
Naples “Federico II”
TASK 2
A NEW HEMICELLULASE (PoAbf) FROM Pleurotus ostreatus
Identification of the enzyme responsible for xylanase activity
produced by P. ostreatus during Solid State Fermentation on
tomato processing waste by means of proteomics analyses
THE BIOCATALYST
THE SUBSTRATES
The corresponding gene and cDNA were cloned and sequenced
(EMBL accession numbers: HE565355 and HE565356,
respectively).
Comparison with other a-arabinofuranosidases indicated that PoAbf
can be classified as a family 51 glycoside hydrolase
Succesful heterologous recombinant expression of PoAbf has been achieved in the yeasts Pichia
pastoris, with a production level of the secreted enzyme of 22*10-3 U mL-1 (Amore et al. 2011)*
rPoAbf has an estimated molecular weight of
81,500 Da, as deduced from SDS-PAGE analyses
O-glycosylation site localized at level of Ser160 as
deduced from mass spectral analyses
rPoAbf exhibits hydrolytic ability against the natural
substrates CM-linear arabinan, wheat arabinoxylan,
arabinotriose and arabinohexaose
PoAbf CATALYTIC PROPERTIES
KM
0.64 ± 0.11 mM
kcat
3,010 ± 145 min−1
Optimal
temperature
40°C
Optimum pH
5
Thermoresistance
pH stability
ENERBIOCHEM PON01_01966
t1/2= 7 days at both 30°C and
40°C
t1/2= 51 days at pH 5
* Amore et al. “Appl Microbiol Biotechnol 2012 May;94(4):995-1006
TASK 2
Design and production of rPoAbf sitedirected mutants
S160G
E364G/E471G
Elucidation of the effect of glycosylation on
enzyme stability
Elucidation of their role in the reaction mechanism
QuikChange® Site-Directed Mutagenesis Kit
S160: unique site of glycosilation;
E364/E471: potential acid/base and nucleophile catalytic residues, respectively
Heterologous recombinant expression in P. pastoris
E364G and E471G mutants show an activity 10-fold and 20-fold lower than
wtPoAbf activity, respectively
The mutant S160G shows a lower thermoresistance than wtPoAbf
ENERBIOCHEM PON01_01966
TASK 2
Development of tailor made enzymes by
directed evolution of PoAbf
Random mutagenesis of poabf cDNA
by Error Prone PCR
Construction of libraries of
randomly mutated forms
of
α-L-arabinofuranosidase
from Pleurotus ostreatus
(PoAbf)*
Construction of libraries by
transformation of the selected
Host, Saccharomyces cerevisiae
(W 303 A1 strain),with
mutated poabf cDNAs
High-throughput screening
of mutants in multi-well plates
based on appropriate criteria (higher activity
and resistance to appropriate operative
conditions than the wild-type)
Characterization of the best (more active and
resistant to appropriate operative conditions
than the wild-type) variants
ENERBIOCHEM PON01_01966
BIOASSORT
www.bioassort-europe.com
Improvement of technologies and tools, e.g. biosystems and biocatalysts, for waste conversion
to develop an assortment of high added value eco-friendly and cost-effective bio-products
Seventh Framework Programme Marie Curie Actions People
International Research Staff Exchange Scheme
The overall aim of this program is to improve technologies and biological tools for conversion of wastes- Organic
Fraction of Municipal Solid Wastes (OFMSW) and agro-industrial wastes.
One of the main objectives is enlarging the patrimony of microbes for waste valorisation by
i)
screening the assortment of thousand microbes belonging to the collections of partners to select the
microbes most efficient in producing enzymes, Ethanol (EtOH), lactic acid (LA), polylactic acid (PLA) and
polyhydroxyalkanoates (PHAs)
ii) discovering novel strains with high potential of production of these bioproducts exploring the biodiversity of
micro flora
iii) exchanging technologies and tools developed by different EU and ICPC partners for genetic engineering of
different microbes (lignocellulolytic microbes, lactic acid bacteria, PHA producers) within the network.
Development of BIOASSORT complex databases on the microbial patrimony
of the network and the main systems, tools and technologies developed.
TASK 1
NEW (HEMI) CELLULOLYTIC MICROORGANISMS*
WP2: Biosystems optimization
i)Enlarging the patrimony of microbes for waste valorisation
ii)Improving ligno(cellulo)lytic abilities, PHA production, optical purity of L-LA by genetic
engineering
Isolation and purification of microorganisms from
50 soil samples (Western ghat regions, Kerala,
INDIA)
Screening of 93 microorganisms for (hemi)cellulolytic
activity production by Congo red assay
Selection of 13 xylanolytic microorganisms and 17
cellulolytic microorganisms
* In collaboration with Prof. Ashok Pandey and Dr. Binod Parameswaran -National Institute for Interdisciplinary
Science and Technology (NIIST ) Council Of Scientific And Industrial Research (Trivandrum, INDIA)
• Selection of the
isolates
considering both
morphology
and soil source
• Set up of liquid
culture in
CMC/XYLAN
containing
media fo,r
enzyme activity
assay
• Identification of
the selected
microorganisms
by 16S rRNA
gene
sequencing
VALORIZATION PROCESS
NEW (HEMI)CELLULOLYTIC MICROORGANISMS
GENOMIC ANALYSES
SELECTION
TASK 1
• Set up of solid
state
fermentation
with at least one
of the selected
microorganisms
(bacteria from
NIIST and/or
fungi from IRDIMEP) on
agroindustrial
wastes
Acknowledgments
Industrial Research Project “Integrated agro-industrial chains with high energy efficiency
for the development of eco-compatible processes of energy and biochemicals production
from renewable sources and for the land valorization- EnerbioChem” PON01_01966 funded
in the frame of Operative National Programme Research and Competitiveness 2007–2013 D.
D. Prot. n. 01/Ric. 18.1.2010.
Research Project funded by European Union in the frame of Seventh Framework Programme
(Marie Curie Actions People International Research Staff Exchange Scheme) 2012-2016
“Improvement of technologies and tools, e.g. biosystems and biocatalysts, for waste
conversion to develop an assortment of high added value eco-friendly and cost-effective bioproducts”-BIOASSORT
ENERBIOCHEM PON01_01966
Acknowledgments
Prof. Olimpia Pepe, Dr Valeria Ventorino
Department of Food Science, University of Naples "Federico II“, Italy
Dr Angela Amoresano, Prof. Leila Birolo,
Department of Chemical Sciences, School of Biotechnological Sciences
University of Naples "Federico II“, Italy
Prof. Bernard Henrissat, CNRS, Universite´s d'Aix-Marseille I & II, Marseille, France
Prof. Sevastianos Roussos, IRD-IMEP Institute, Université Paul Cézanne, Aix Marseille III
Antonella Amore, Simona Giacobbe, Annabel Serpico,
Rossana Liguori , Simona Russo, Tiziana Russo
Vincenza Faraco
Department of Chemical Sciences (DSC)
School of Biotechnological Sciences
Università Degli Studi Di Napoli “Federico II”
E-mail address vfaraco@unina.it
phone + 39 081674315/679895
mobile phone +39 3403483751
fax +39 081674313
THANKS FOR YOUR
ATTENTION