Università Degli Studi Di Napoli “Federico II” ENERBIOCHEM PON 01_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, 2 D 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” PON 01_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 WP 3 (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; My. Cycler 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 LC -MS 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 (4 x 500 ml, 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 Fiber. Cap™ 2021/2023 (Foss) for analyses of lignin, cellulose and hemicellulose contents, fibres and ash.
2 nd Generation Bioethanol It is produced from lignocellulose biomass, differently from 1 st 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
2 nd 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 C 5/C 6 -Sugars Fermentation
Main challenges. . …. . to increase 2 nd 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 p. H 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. (1 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/m. L of AZO-CMCase activity) Microorganisms identificatiom (DNA fingerprinting and 16 S r. RNA gene sequencing) ENERBIOCHEM PON 01_01966 in collaboration with Prof. Olimpia Pepe, University of Naples “Federico II”
TASK 2 Identification and characterization of new glycosyl hydrolases Bacillus amyloliquefaciens sp. B 31 C Streptomyces sp. G 12 q Zymogram analyses Streptomyces sp. G 12 Bacillus sp. B 31 C q 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. B 31 C Coning and recombinant expression of the gene codifying for Streptomyces sp. G 12 glycosyl hydrolase cellstrep in Escherichia coli, characterization of the recombinant enzyme. Cellulose binding protein from Streptomyces griseoflavus ENERBIOCHEM PON 01_01966 * in collaboration with Prof. Leila Birolo, DSC, University of Naples “Federico II”
TASK 2 A NEW HEMICELLULASE (Po. Abf) 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 c. DNA were cloned and sequenced (EMBL accession numbers: HE 565355 and HE 565356, respectively). Comparison with other a-arabinofuranosidases indicated that Po. Abf can be classified as a family 51 glycoside hydrolase Succesful heterologous recombinant expression of Po. Abf has been achieved in the yeasts Pichia pastoris, with a production level of the secreted enzyme of 22*10 -3 U m. L-1 (Amore et al. 2011)* r. Po. Abf has an estimated molecular weight of 81, 500 Da, as deduced from SDS-PAGE analyses Po. Abf CATALYTIC PROPERTIES KM O-glycosylation site localized at level of Ser 160 as deduced from mass spectral analyses r. Po. Abf exhibits hydrolytic ability against the natural substrates CM-linear arabinan, wheat arabinoxylan, arabinotriose and arabinohexaose 0. 64 ± 0. 11 m. M kcat 3, 010 ± 145 min− 1 Optimal temperature 40°C Optimum p. H 5 Thermoresistance p. H stability ENERBIOCHEM PON 01_01966 t 1/2= 7 days at both 30°C and 40°C t 1/2= 51 days at p. H 5 * Amore et al. “Appl Microbiol Biotechnol 2012 May; 94(4): 995 -1006
TASK 2 Design and production of r. Po. Abf sitedirected mutants S 160 G E 364 G/E 471 G Elucidation of the effect of glycosylation on enzyme stability Elucidation of their role in the reaction mechanism Quik. Change® Site-Directed Mutagenesis Kit S 160: unique site of glycosilation; E 364/E 471: potential acid/base and nucleophile catalytic residues, respectively Heterologous recombinant expression in P. pastoris E 364 G and E 471 G mutants show an activity 10 -fold and 20 -fold lower than wt. Po. Abf activity, respectively The mutant S 160 G shows a lower thermoresistance than wt. Po. Abf ENERBIOCHEM PON 01_01966
TASK 2 Development of tailor made enzymes by directed evolution of Po. Abf Random mutagenesis of poabf c. DNA by Error Prone PCR Construction of libraries of randomly mutated forms of α-L-arabinofuranosidase from Pleurotus ostreatus (Po. Abf)* Construction of libraries by transformation of the selected Host, Saccharomyces cerevisiae (W 303 A 1 strain), with mutated poabf c. DNAs 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 PON 01_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 (Et. OH), 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* WP 2: 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 16 S r. RNA 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- Enerbio. Chem” PON 01_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 PON 01_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
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