Prospecting Hydrolases of microorganisms and finding their potential for bioenergy production

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1 LABORATORY OF MICROBIOLOGY AND CELLULAR BIOLOGY Prospecting Hydrolases of microorganisms and finding their potential for bioenergy production Maria de Lourdes T. M. Polizeli Emanuelle Corbi Corrêa USP São Paulo University FFCLRP Faculty of Philosophy, Sciences and Letters of Ribeirão Preto Biology Department

2 Presentation outline Biomass Biofuels Enzymes from microorganisms Perspectives

3 Biomass Biomass What is it? All renewable resources which come from organic matter and that can be used to produce energy. Three domains of life and their wastes tons Source: Fair Biomass Brazil

4 Biofuels Biofuels Solid, liquid or gaseous fuel wood, grass cuttings, domestic refuse, charcoal, dried manure, residues Vegetable oil, alcohol, biodisel Syngas, Biogas Advantages

5 Biofuels ENERGY GONE

6 Palm Coconut Soy Cotton Sunflower Seeds Sugarcane Corn Eucalyptus Rice Coffee Brazil Energy Matrix Input SUPERIOR ENRGY BALANCE Fonte: World Watch Institute (2005) and Macedo et al. (2008)

8 BRAZIL: EXPANSION POTENTIAL BRAZIL IS THE ONLY COUNTRY IN THE WORLD WITH GREAT CAPACITY TO EXPAND ITS AGRICULTURAL PRODUCTION (with sustentability) Expansion area in the Brazilian Cerrado region (million hectares) Total Area Area good for agriculture Area in use for cattle raising... (35) Occupied area (forests & plantations) (12) Available Area Source: EMBRAPA

9 Second generation ethanol sugar Sugarcane 426 million tons alcohol alcohol animal food electricity m3 Fonte: CONAB

10 Ethanol It is biodegradable, made from renewable sources Low toxicity to aquatic and soil forms of life Virtually no sulfur and particulate emissions Reduces emissions of CO and toxic substances Blended with other fuels Manufacture biodisel (methanol) Source of hydrogen (fuel cells) Increasing Market of flexible fuel vehicles Alternative fuel for motorcycles, boats, airplanes Plastic production Electricity

11 Cellulosic ethanol tons tons tons tons L Fonte: UNICA

12 Polizeli (2008). Current Advances in Fungal Biotechnology. Cell wall structure

Enzymes: Amylases Celulases Xylanases Xylosidases Ligninases Invertases Lipases Pectinases Aspergillus

rhizopodiformis; Paecilomyces variotii Aspergillus phoenicis, A. niveus; A. japonicus; A. ochraceus; A.

niger; A. terricola; Malbranchea puchella Aspergillus phoenicis; A.

niger; Cordyceps brongniartii;penicillium purpurogenum Aspergillus terreus; A.

rhizopodiformis; Paecilomyces variotii Acid and alkaline phosphatases Phytases Aspergillus niger; A.

13 Enzymes: Amylases Celulases Xylanases Xylosidases Ligninases Invertases Lipases Pectinases Aspergillus phoenicis, A. niveus; Rhizopus microsporus var. rhizopodiformis; Paecilomyces variotii Aspergillus phoenicis, A. niveus; A. japonicus; A. ochraceus; A. niger; A. terricola; Malbranchea puchella Aspergillus phoenicis, A. niveus; A. japonicus; A. ochraceus; A. niger; A. terricola; Malbranchea puchella Aspergillus phoenicis, A. niveus; A. japonicus; A. ochraceus; A. niger; A. terricola; Malbranchea puchella Aspergillus phoenicis, A. niveus; A. japonicus; A. ochraceus; A. niger; A. terricola; Malbranchea puchella Aspergillus phoenicis; A. caespitosus Trichoderma pseudokoningii; A. caespitosus; A. niger; Cordyceps brongniartii;penicillium purpurogenum Aspergillus terreus; A. niveus; Rhizopus microsporus var. rhizopodiformis; Paecilomyces variotii Acid and alkaline phosphatases Phytases Aspergillus niger; A. niveus; A. ochraceus. Aspergillus niger; A. niveus; A. ochraceus. Syrup starch Cheese preparation Fruit juice Pulp & paper Animal food detergent Ethanol

14 Polizeli, M.L.T.M. Properties and Commercial Applications of Xylanases from fungi. In: Mycotechnology: Current Trends and Future Prospects, I.K. Int. Publisher, 4: 82108, BIOBLEACHING OF CELLULOSIC PULP A B C D a b c d e f g h (f) NaOH/O 3 /ClO 2 (g) NaOH/O 2 /H 2 O 2 (h) NaOH/ClO 2

15 Angiosperms Gimnosperms Polizeli, M.L.T.M. et al., Xylanases from fungi: properties and industrial applications. Review. Appl. Microbiol. Biotechnol., 67, , 2005

16 Filamentous fungi Polizeli, M.L.T.M. et al., Xylanases from fungi: properties and industrial applications. Review. Appl. Microbiol. Biotechnol., 67, , 2005

17 Collection of samples beehive Soil and leaves Sugar cane soil edge of the river Soil and humus of farm Thermal water Orange trees soil Eucalipto forest Maize plantation

18 Fungus samples

19 Fungus isolates

20 Fungi storaging

21 Fungi characterization 1 m 1 m

22 Fungi characterization

23 Enzymatic Synthesis Induction Catabolite Repression xylosidase Xilobiose Xilotriose Xilooligosaccharides Permeases Xylose Glucose Cytoplasm Plasma Membrane Xilobiose Xilotriose Xilooligosaccharides Glucose xylanase Xylan Polizeli, M.L.T.M. et al., Xylanases from fungi: properties and industrial application. In: Appl Microbiol Biotechnol 67: , 2005.

24 Effect of carbon sources on ßxylosidase and xylanase production Polizeli, M.L.T.M. et al., J. Ind. Microb. & Biotechnol. 26, , Rizzatti, A. C. et al., J. Ind. Microbiol. Biotechnol. 35 (4), ,.2008.

supplemented with 1% glucose, during 72 h, at 42 C and transferred to the

25 Effect of glucose, dibutyrylcamp or camp on xylanase production Extracellular activity Mycelial extract The fungus was previously grown on Vogel medium supplemented with 1% glucose, during 72 h, at 42 C and transferred to the different media during 6 h. N = 4. Polizeli, M.L.T.M. J. Ind. Microbiol. & Biotechnol. 35: , 2008.

26 0.1% xylan 0.1% glucose 0.1% xylose 100 μm camp 100 μm dcamp Northern blot hybridization of the A. phoenicis gene ApXLN Polizeli, M.L.T.M. J. Ind. Microbiol. & Biotechnol. 35: , 2008.

27 Growth (Total protein, mg) Spore germination of A. phoenicis at 25 C (A) and 42 C (B) for 8 hs 20 C Hours (C) Growth at 25 C ( ) and 42 C ( ). Polizeli, M.L.T.M. et al., J. Ind. Microbiol. & Biotechnol. 31, 8893, 2004.

28 Xylosidase activity (U/mg prot) Xylanase activity (U/mg prot) Timecourse of xylosidase (A) and xylanase (B) production Extra A B C intra C Hours Polizeli, M.L.T.M. et al. J. Ind. Microbiol. & Biotechnol. 31, 8893, 2004.

29 Percent activity Effect of temperature on activities 100 xylosidase A xylanase C 80 extracellular B D intracellular Temperature (ºC) C (, ), 42 C (, ) Polizeli, M.L.T.M. J. Ind. Microbiol. & Biotechnol. 31, 8893, 2004.

30 Xylosidase activity (%) Xylanase activity (%) Thermal inactivation 100 A B C 60 C Time (minutes) 25 C (, ); 42 C (, ); extra ( ), intra ( ) Polizeli, M.L.T.M. et al. J. Ind. Microbiol. & Biotechnol. 31, 8893, 2004.

31 ELECTRONIC MICROSCOPY OF CELLULOSE TREATED WITH XYLANASE control 2 hours of incubation Xylanase 15 U/g dry cellulose pulp Polizeli, M.L.T.M. et al. submitted.

32 PHYLOGENETIC RELATIONSHIPS AMONG GENUS Aspergillus Corrêa, E. C. et al. (submmited to Molecular Biology and evolution) 2008.

33 Perspectives Development of Enzymes for Biomass Saccharification Bioprospection of filamentous fungi Sparse matrix techniques for optimization of microbial enzyme production OUR LABORATORY Overexpression of enzymes in heterologous organisms Production of enzymes and fungi quick adaptation (directed anagenetic evolution) Comparative and evolutionary approaches of different groups of enzymes Submerged and solidsubstrate fermentations (using residues from agrofarming and forestry industries) catabolic induction and repression studies using diverse carbon sources purification and biochemical characterization of microbial enzymes and sequencing

34 PLAN Fermentation Bioprospection Enzymes cdna Libraries Protein sequencing MALDITOF Edman deg Gene cloning Sequencing of cdna Glicosylome Catalysis Kinetic (Vmax, Km Kcat, etc) Specificity (substrate/ product) Studies of biophysics Spectroscopy (CD, Fluorecense, DLS, FTIR, etc) Expression Systems E. coli yeasts Directed Evolution XRays Cristalography

35 Research Group

Table of Contents 1. CHAPTER I INTRODUCTION 1 [A] GLUCOSE ISOMERASE 1

Table of Contents 1. CHAPTER I INTRODUCTION 1 [A] GLUCOSE ISOMERASE 1 1.1 Enzymatic versus chemical isomerization 4 1.2 Glucose Isomerase producing microorganisms 5 1.3 Structure of Glucose Isomerase 8