Recalcitrância da biomassa e ação enzimática

Transcription

1 Recalcitrância da biomassa e ação enzimática The origin of cell wall recalcitrance and the use of glycosyl hydrolases from basidiomycetes to help on enzymatic saccharification processes André Ferraz Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo Lorena, SP, Brasil Co-autores envolvidos nos trabalhos: Thales H. Costa, Germano Siqueira, Fernando Masarin, Fernanda M Mendes, Débora Laurito-Friend, Gerard Koch, Henrich Scheller, Adriane MF Milagres

2 Introduction - Recalcitrance in lignocellulosic materials Cellulose is recalcitrant by itself (but cellulose is not "clean" as shown in this figure) Lignin and hemicelluloses involve the cellulose nonofibrils in the cell walls causing the primary recalcitrance Cell wall SEM Cell wall models

3 Cellulose conversion (%) Sugar cane as a model lignocellulosic material Sugar cane hybrids selected for contrasting lignin contents Varied recalcitrance was related with the original lignin contents (enzymatic hydrolysis of non-pretreated samples) Hydrolysis time (h) Hybrids provided by Prof. M.H.P. Barbosa. Universidade Federal de Viçosa. MG. Brasil a mill bagasse 14 reference cultivar low lignin mid lignin high lignin Masarin et al. Biotechnol Biofuels 4:

4 Chemical composition of sugar cane hybrids ranked by their lignin contents Clone Total lignin Hemicellulose Glucan Extractives Sum Mill bagasse Reference cultivar Masarin et al. Biotechnol Biofuels 4:

5 Variable recalcitrance in different cells and tissues Cell anatomy in sugar cane vascular bundles Rind Pith

6 Experimental approach % (dry weight basis) % 5-63 % 18-2 % 6-7 %

7 Cellulose conversion (%) Enzymatic hydrolysis of varied sugar cane regions from the internodes Outermost fraction Rind Interface Pith 8 Hybrid 58 6 Pith Interface pith-rind Rind Outermost fraction Enzymatic hydrolysis: 1 FPU/g; 2 IU - 45 o C Hydrolysis time (h) Costa et al. Ind Crops Prod 51:

8 Enzymatic hydrolysis of varied fractions from lignincontrasting hybrids Outermost fraction Rind Interface Pith Pith Interface pith-rind Rind Outermost fraction Hybrid 14 lignin lignin Hybrid 58 Hybrid 89

9 Chemical composition of each region in the sugar cane internodes Samples Internode region Extractives Glucan Hemicellulose Total lignin H14 Pith Interface Rind Outermost fraction H58 Pith Interface Rind Outermost fraction H89 Pith Interface Rind Outermost fraction Costa et al. Ind Crops Prod 51:

10 Cellulose conversion after 72h (%) Correlation studies Enzymatic hydrolysis versus area occupied by the vascular bundles hybrid 14 hybrid 58 hybrid 89 pith of the H % of the area occupied by vascular bundles rind of the H14 Costa et al. Ind Crops Prod 51:

11 The role of lignin in the recalcitrance (UV-microspectrophotometry was used to map lignin and hydroxycinnamic acids in the cell walls from these regions) Example of a selected point (1 µm 2 ) to record the UV spectrum 4 x

12 Absorbance Absorbance Average spectrum from different cells in sugar cane Rind vessel fiber parenchyma Pith vessel fiber parenchyma Wavelenght (nm) Wavelenght (nm) 28 nm > aromatic ring substituted with oxygenated groups 315 nm > aromatic ring conjugated with alfa-carbonyl or alfabeta unsaturated groups >>> in grasses correspond to ferulic and coumaric acids Siqueira et al. Biotechnol Biofuels 4:7. 211

13 Mapping lignin in the different regions of the sugar cane hybrids Vessel Fiber parenchyma rind Hybrid 14 Hybrid 58 Hybrid interface pith Costa et al. Ind Crops Prod 51:

14 Correlation studies Predicting parameter for cellulose conversion glucan/(lignin + hemicellulose) Costa et al. Ind Crops Prod 51: large area example small area example

15 Non-cellulosic polysaccharides Hydrolysis with 15% trifluoracetic acid under mild conditions Sample Internode region Chemical composition (%. w/w) Xylans Arabinosyl Glucans Galactans Galacturonic acid H 58 Pith Interface Rind H 89 Pith Interface Rind H 14 Pith Interface Rind Xylans should prevails in sugar cane samples Glucans were high, especially in non-recalcitrant pith fractions Which non-cellulosic glucans are in pith fractions?

16 Mixed ( )-ß-glucans as candidate hemicelluloses Immune Dot blots for mixed linkage glucans 58 Pith Inter Rind KOH 4M / NaBH 4 extracts Monoclonal antibody for (1-3,1-4)-ß-glucan: MOUSE IgG 89 14

17 Mixed glucans (% w/w) Lichenase-based determination of the mixed linkage glucans H89 H58 H14 Pith Pith-rind interface Rind

18 Immunohistochemistry detection of hemicelluloses - Mixed-linkage ( )-ß-glucan: primary monoclonal antibody (MOUSE IgG); secondary antibody: Alexa Fluor Xylan: primary monoclonal antibody (RAT IgG) LM1; secondary antibody: Alexa Fluor Arabinoxylan: primary monoclonal antibody (RAT IgG) LM11; secondary antibody: Alexa Fluor 568 Fluorophore secondary primary Collaborative work with HV Scheller, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

19 Immunohistochemistry detection Mixed linkage glucans light intensity is proportional to the content Pith Hybrid 58 Magnif. 5x Interface Rind

20 Xylans in the sugar cane fractions LM1: antibody for xylan LM11: antibody for arabinoxylan light intensity is proportional to the content controls without antibody xylan arabinoxylan

21 Correlation studies Predicting parameter for celullose conversion glucan/(lignin + hemicellulose) cellulose + MLG mainly xylan large area example small area example

22 Lignin content (16.8 to 21.5) Technology derived data Process integration and breeding benefits Hybrid number Plant productivity (wet ton/ha) Internodes diameter (cm) Plant bending (*) Dry biomass content (bagasse) (kg/ton of wet plant) Sucrose yield (kg/ton of wet plant) (**) Degree of plant bending varying from straight (1) to bended (5) stalks Masarin et al. Biotechnol Biofuels 4:

23 Technology derived data Process integration and breeding benefits Glucose yields from alkaline-sulfite pretreated materials hydrolyzed with commercial cellulases Laurito-Firiend el at. Industrial Crops and Products 75: 65-75, 215

24 Recent attempts to increase polysaccharide hydrolysis efficiency - a multidisciplinar approach Searching glycoside hydrolases from wood decay fungi as supplemental proteins for hydrolysis of pretreated sugar cane bagasse Fernanda Valadares, Thiago Gonçalves, Dayelle SPO Gonçalves, Fernando Segato, Elisson Romanel, Adriane MF Milagres, Fábio Squina, André Ferraz

25 Enzymatic activities of glucoside hydrolases in Laetiporus sulfureus and Pleurotus ostreatus extracts and in reference commercial enzymatic preparations. Enzyme source Relative hydrolytic activities in the protein extracts (calculated with basis on IU. mg -1 protein and β-glucosidase activity set to the unit) FPAase Endoglucanses Cellobiohydrolases β-glucosidases Xylanases β-xylosidase L. sulfureus nd* P. ostreatus nd* 1. nd* T. reesei A. niger nd* nd* nd* T. emersonii nd 61 nd nd 44 nd

26 Cellulose conversion (%) Xylan conversion (%) Effect of enzyme loading on the hydrolysis of alkalinesulfite pretreated sugar cane bagasse - use of commercial enzymes 1 a 1 b T. reesei enzymes (1% load) T. reesei enzymes (5% load) T. reesei enzymes (25% load) Hydrolysis time (h) Hydrolysis time (h) 1% load = 1 FPU and 15 IU -glycosidase/g of substrate

27 Cellulose conversion (%) Xylan conversion (%) Adding basidiomycetes enzymes to restore total endoglucanase activities on the hydrolysis of pretreated sugar cane bagasse 1 a 1 b T. reesei (1% load) T. reesei (5% load) T. reesei (5%) + L. sulfureus (5%) T.reesei (5%) + P. ostreatus (5%) Hydrolysis time (h) Hydrolysis time (h)

28 Initial reaction rates during enzymatic hydrolysis of alkaline sulfite pretreated sugar cane bagasse by several enzyme mixtures Enzyme mixture used in the hydrolysis experiment Initial hydrolysis rate (%.h -1 ) Cellulose Xylan T. reesei 1% (1 FPU/g) T. reesei 5% (5 FPU/g) T. reesei 25% (2.5 FPU/g)) T. reesei (5%) + L. sulfureus (5%) T. reesei (5%) + P. ostreatus (5%) T. reesei (5%) + T. emersonii (5%)

29 LC/MS/MS identification of proteins in the basidiomycetes extracts - genome database analysis n t = 45 n t = 13 GH45-EG in L sulf GH3 -glyc in both cloning and expression in ascomycetes is under way...

30 Conclusions - In sugar cane, the recalcitrance was variable in different regions of the internode >> pith was very low in recalcitrance whereas the rind and the outermost fraction were very recalcitrant - Low recalcitrance in the pith region was associated with the predominance of parenchyma cells that contained less aromatic compounds (lignin and hydroxycinnamic acids) and the presence of mixed linkage glucans - There was a significant variation in the recalcitrance of the sugar cane hybrids selected for contrasting lignin contents >> low lignin contents or more available glucan resulted in increased efficiency in the cellulose hydrolysis by enzymes - Sugar cane evaluation can help the breeders or the researchers looking for less recalcitrant cell walls to select plants with low lignin content or even plants expressing mixed linkage glucans into the cell walls. - Basidiomycetes merits exploitation for new glycoside hydrolases

31 Acknowledgements Financial support from FAPESP (8/ ; 11/5535-2; 14/6923-6), CNPq and CAPES

32 Mixed linkage glucans inside a vascular bundle light intensity is proportional to the content Hybrid 58 Magnif. 2x