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Bio Catalysts
- 1. Bio-catalysts in Industrial and Environmental biotechnology – microorganisms – enzymes Selection and improvement Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 2. Microorganisms • Where are they found? • How diverse are they? • Role in geochemical nutrient cycles. • How do they grow and what are their requirements for growth and biodegradation? Microorganisms in waste treatment: • Biodegradation and environmental clean up. • Microbial production and products in industry Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 3. Microorganisms in the environment; Challenging conventional views of life. • Sagan and Margulis (1998) “Garden of Microbial Delights”. – “ALL of the elements crucial to global life- oxygen, nitrogen,phosphorus, sulfur, carbon- return to a usable form through the intervention of microbes… Ecology is based on the restorative decomposition of microbes and molds, acting on plants and animals after they have died to return their valuable chemical nutrients to the total living system of life on earth” • Gould (1996) “Life’s Grandeur” The Power of the Modal Bacter. – The first multicellular organisms do not enter the fossil record until about 580 million years ago - this is after about five sixths of life’s histroy have passed. Bacteria have been the stayers and keepers of life’s history. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 4. Microorganisms in the environment; Challenging conventional views of life. •Sagan and Margulis (1998) “Garden of Microbial Delights”. –“ALL of the elements crucial to global life- oxygen, nitrogen,phosphorus, sulfur, carbon- return to a usable form through the intervention of microbes… Ecology is based on the restorative decomposition of microbes and molds, acting on plants and animals after they have died to return their valuable chemical nutrients to the total living system of life on earth” •Gould (1996) “Life’s Grandeur” The Power of the Modal Bacter. –The first multicellular organisms do not enter the fossil record until about 580 million years ago - this is after about five sixths of life’s histroy have passed. Bacteria have been the stayers and keepers of life’s history. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 5. Microorganisms Where are they found? Diverse environments • Virtually every environmental niche • Extremes of pH and salinity • Extremes of temperature and pressure • Without air (Anaerobic) • Growth on many chemical substrates • Attached to surfaces in biofilms • Geothermal vents and subterranean deposits Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 6. Microorganisms Where are they found? Biomass on the planet. • Most culturing analysis misses over 99% of the microbial population. • Molecular techniques now reveal hidden diversity • Heterotrophs - 5-20% biomass in sea waters - up to 80% of the primary production • Rich bacterial communities in sub-surface strata (600 m deep) - up to 2 x 104 tons - more than all flora and fauna -equivalent to 2 m layer over planet! Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 7. Microorganisms How diverse are they? Plants & Eubacteria Animals Archaea • Diverse range of species • Earliest life on the planet • Anaerobic then aerobic • Three Kingdoms • Eukaryote Plants & Animals • Eubacteria • Archaebacteria • Exteme living bacteria 3 billion years Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 8. Microorganisms How diverse are they? Diversity of bacteria in soil 16s rRNA sequences reveal true diversity in soil DNA Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 9. Microorganisms Role in geochemical nutrient cycles. • Microorganisms play a role as: •PRIMARY PRODUCERS •BIODEGRADERS AND CONSUMERS • Critical role in cycles of many elements; • Carbon and and Oxygen cycle • Nitrogen cycle • Sulfur cycle • Phosphorus cycle Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 10. Microorganisms How do they grow: requirements for biodegradation? • Nutrients • Carbon, Nitrogen, Phosphorus, Sulfur • Many chemicals supply these • Micronutrients/ trace metals/ vitamins • Electron acceptors - usually O2 • Converts / burns carbon substrate to CO2 Energy and biomass ie GROWTH Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 11. Microorganisms Biodegradation O2 consumption GROWTH - CELL DIVISION 2.0µm INCREASE IN BIOMASS ORGANIC POLLUTANT AND NUTRIENTS (C,P,N,O,Fe,S……) SINGLE CO2 BACTERIUM evolved Controlled release of energy Slow Burning! Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 12. Microorganisms Oxygen and Electron Acceptors: crucial for Biodegradation reactions in the environment. 2H+ H2 O O2 SUBSTRATE ADP METABOLISM Pi ATP H2/2e- CARBON ENERGY GROWTH/Biomass CO2 Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 13. Microorganisms Role of electron acceptors; rate of biodegradation O2 NO3- SO42- Fe3+ H2O NO2- H2S Fe2+ N2 0.814V -0.214V -0.185V 0.741V FAST SLOW GROWTH GROWTH Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 14. Microorganisms Anaerobic growth and biodegradation Fermented Organic matter Acetic Acid + H2 , CO2 Methanogenesis CH4 , CO2 , H2O Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 15. Microorganisms Fixation of oxygen as a first step in biodegradation Cell membrane ReductaseNAP FerredoxinNAP ISPNAP NAD+ (OX) (OX) (OX) O2 OH OH NADH ReductaseNAP FerredoxinNAP ISPNAP + H+ (RED) (RED) (RED) Cell Biomass Further degradation CO2 Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 16. Microorganisms Biological waste treatment; Managing microorganisms for environmental cleanup • 10 x 106 Chemicals – 8 x 106 Xenobiotic – 1 x 106 Recalcitrant • 0.4 x 106 traded at over 50 tonnes per year • Toxicological/ biodegradative data on only around 5000-6000 •Municipal waste-water treatment •Biodegradation of industrial wastes • petrochemicals, bulk chemical processes • textiles, leathers • metals • Remediation of contaminated land in situ Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 17. Microorganisms Biological waste-water treatment: The activated sludge process. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 18. Microorganisms Biological waste treatment; Advanced industrial membrane reactor. E F F LU E N T F R E E O F P O LLU TA N T W A S T E - W A T E R C O N T A IN IN G P O L L U T A N T S Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 19. Microorganisms Cultivation of microorganisms for industrial use. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 20. Microorganisms Cultivation of microorganisms for industrial use. Advanced laboratory fermenters Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 21. Products from Microorganisms: Overview of range of examples. • Various foods and drinks • Enzymes for varied uses (GM enzymes); biocatalysts • Engineered proteins ( antibodies ) • Vaccines and antibiotics (secondary metabolites) • Primary metabolites and bulk chemicals (amino acids (glutamic acid) and organic acids (acetic acid) • Pharmaceuticals and novel chiral chemicals • Recovery of metals in bioleaching • Biosensors (use of enzymes to specifically detect chemicals in medical and ) Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 22. Enzymes Protein engineering Tailor-made biocatalysts • The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity • Rational (re)design versus directed evolution Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 23. Enzymes Protein engineering Genetic manipulation techniques • Large-scale supply of enzymes at reasonable price • Identification of new biocatalysts (screening) doesnot always yield suitable enzymes for a given synthetic problem • Computer-aided site-directed mutagenesis • Directed (molecular) evolution Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 24. Enzymes Protein engineering Site-directed mutagenesis • Requires structural information and knowledge about relationship between sequence, structure, function and mechanism • Very information-intensive • Rapid progress in NMR / X-ray methods • Genome sequence information • Molecular modeling, bioinformatics • Prediction of selectivity, activity, stability etc. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 25. Enzymes Protein engineering Rational redesign strategy • Protein structure • Planning of mutants, SDM • Vectors containing mutated genes • Transformation in E. coli • Protein expression and purification • Mutant enzyme analysis • Negative mutants • Improved mutant enzymes Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 26. Enzymes Protein engineering Rational redesign • Amino acid substitutions often selected by sequence comparison with homologous sequences • Results have to be carefully interpreted • Minor changes by a single point mutation may cause significant structural disturbance • Comparison of 3D-structure of mutant and wild- type enzyme necessary Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 27. Enzymes Protein engineering Directed evolution • Evolutive biotechnology, molecular evolution • Random mutagenesis of the gene encoding the biocatalyst (e.g. by error-prone PCR) • DNA shuffling: recombination of gene fragments (staggered extension process or random priming recombination) Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 28. Enzymes Protein engineering Directed evolution strategy • Random mutagenesis • Library of mutated genes • Transformation in E. coli • Mutant library > 10.000 clones • Protein expression in microtiter plates • Selection parameters • Mutant enzyme and product analysis • In vitro-recombination, transformation etc. Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 29. Enzymes Protein engineering Selection parameters • Substrate range • Stability in organic solvent • Stability towards reaction conditions • Thermal stability • High-throughput product analysis • Robot technology Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 30. Enzymes Protein engineering Selection parameters • Hydrolysis of esters: agar-plate assay based on pH indicators • Parallel assaying of replica-plated colonies with substrate analog • Isotopically labeled substrates • Capillary electrophoresis (7000 samples per day) • Optimization with saturation mutagenesis Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica
- 31. Enzymes Protein engineering Improving thermostability • Cold-adapted proteases • Combined screening for activity, thermostability, organic solvent tolerance and pH-profile • Engineering of entire metabolic pathways • Phytoene desaturase and lycopene cyclase shuffling for carotenoid biosynthesis • Molecular breeding Giovanni Sannia Università degli Studi di Napoli Federico II Dipartimento di Chimica Organica e Biochimica