Biofiles V5 N5

Biofiles Volume 5, Number 5

Enzymes and Reagents for Alternative Energy Metabolic strategies in alternative energy

Enzyme-based fuel cells

Lignocellulosic depolymerization

Enzymes for biofuel research

Reagents for ethanol and biodiesel analysis Biofilesonline Biofilescontents Your gateway to Biochemicals and Reagents for Life Science Research Introduction 3

BioFiles Online allows you to: Advancements in Enzyme-Based • Easily navigate the content of Fuel Cell Batteries, Sensors and the current BioFiles issue Emissions Reduction Strategies 8 • Access any issue of BioFiles • Subscribe for email notifications Selected Enzymes for Biofuel of future eBioFiles issues Cell Research 10 Register today for upcoming issues and eBioFiles announcements at Enzymes and Reagents for sigma.com/biofiles Ethanol Research 12 Enzymes for Lignocellulosic Greener Products Ethanol Research 12 Sigma-Aldrich is committed to doing our Enzymes for Starch Hydrolysis 20 part to minimize our footprint on the Ethanol Analysis 23 environment. As a life science and high technology company, we recognize your Enzymes for BioDiesel Research 24 need for high quality chemicals comes Lipase 24 first. In cases where it becomes possible to consider alternatives, we have made it Phospholipase C 24 easier for you to find greener options BioDiesel Analysis 25 through our new “Greener Alternatives and Technologies” product lists. Metabolic Standards for Mevalonate and Isoprenoid Pathway Analysis 30 Products Including: • Quality Environmentally-Friendly Enzymes • Ionic Liquids • Greener Alternatives: Solvents • Greener Alternatives: Reagents sigma-aldrich.com/green

Life Science Labware Center The Life Science Labware Center allows easy browsing of labware products, including disposables, books, and equipment

Features Include: • Corning® cell culture selection guide • Products by top manufacturers • New products by research areas • New lab start-up programs Start using the Life Science Labware center at sigma-aldrich.com/lslabware Technical content: Robert Gates, M. S. M. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 3

Introduction

Robert Gates Market Segment Manager [email protected]

Leveraging nature’s metabolic glucose, and then needlessly polymerize strategies for tomorrow’s this glucose into starch or cellulose. Then energy resources. we often rely on microbial and enzymatic hydrolysis of these biopolymers to give back Although most of the world’s population glucose and then ferment it to yield ethanol. understands that carbon dioxide is the Sugarcane ethanol employs a slightly simpler predominant end product of energy path via sucrose. production from combustible fuels, most of us forget that the “raw material” for most Our challenge for the future may be to find of our combustible fuel sources is also the “straightest metabolic line” between atmospheric carbon dioxide. Photosynthetic carbon dioxide and ethanol, butanol, fatty carbon dioxide fixation is most often the first acids or even hydrogen. The ideal route to step in the synthesis of both our renewable ethanol production might be to devise an fuels and non-renewable fossil fuels. Even enzymatic or cellular system to fix carbon much of our hydrogen production is an dioxide and take the direct route from indirect product of carbon dioxide fixation as ribose-1,5-bis-phosphate to pyruvate to it is often derived from fossil fuels. ethanol and bypass glucose biosynthesis, polymerization and depolymerization. The basic differences between non- We have included the Nicholson/IUBMB renewable fossil fuels and our “new Metabolic Pathway chart on the following generation” renewable fuels is time, energy pages illustrating the specific pathways input and mass. Our non-renewable related to carbon dioxide fixation and fuels, such as petroleum and coal required glucose, cellulose, pectin, starch, lignin and thousands of years of collection of solar ethanol production highlighted in yellow. energy to power carbon fixation for the biosynthesis of enormous amounts of Enzymes and their metabolic pathways biomass. That biomass then underwent enter the energy equation from other millions of years of anaerobic decomposition directions. Microbial strains are being to yield today’s fossil fuels. In our attempt to discovered that can ferment cellulosic circumvent or speed up this process, we are biomass to yield hydrogen. Some finding natural “renewable analogs” to some photosynthetic microbes, such as of our fossil fuels, i.e., ethanol and butanol algae, are capable of splitting water to to replace or supplement gasoline and fatty produce hydrogen utilizing solar energy. acid esters to replace diesel. Hydrogenases are being targeted not only for hydrogen production but also for the However, we have not advanced far enough oxidation of H2 in fuel cells. Immobilized in our manufacturing technology to divest enzyme-based fuel cell batteries can ourselves of the need for biological systems utilize multi-enzyme systems that mimic to do the complicated work for us. Current in vivo glycolytic and Krebs Cycle pathways. processes for ethanol production rely on Immobilized enzymes are also being plants to fix carbon dioxide, synthesize studied for the sequestration of carbon a “common currency” molecule such as dioxide emissions. 4

Engineering organisms and enzyme systems below, many of these areas require an (5) D. Klein-Marcuschamer, V. G. Yadav, A. Ghaderi, G. Stephanopoulos, De Novo Metabolic Engineering and to maximize biofuel production could understanding of metabolic pathways and the Promise of Synthetic DNA, Adv. Biochem. Eng. Biotech- require insertion of exogenous enzymatic enzymatic processes. nol. in press (2010). pathways from various organisms into a (6) J. L. Fortman, S. Chhabra, A. Mukhopadhyay, H. Chou, T. S. References Lee, E. Steen, J. D. Keasling, Biofuel alternatives to etha- new host. In addition, alternative metabolic nol: pumping the microbial well, Trends in Biotechnol., 26 (1) S. Atsumi, J. C. Liao, Metabolic Engineering for Ad- 375–381 (2010). pathways such as the mevalonate and vanced Biofuels Production from Escherichia coli, Curr. (7) D. B. Levin, R. Islam, N. Cicek, R. Sparling, Hydrogen Opin. Biotechnol., 19, 414–419 (2008). isoprenoid pathways are being studied as production by Clostridium thermocellum 27405 from (2) H. Alper, G. Stephanopoulos, Engineering for biofuels: cellulosic biomass substrates., Int. J. Hydrogen Energy, sources of biofuel. exploiting innate microbial capacity or importing 31, 1496–1503 (2006) biosynthetic potential?, Nature Reviews, Microbiology, 7, This issue of BioFiles is devoted to how (8) M. L. Ghirardi, S. Kosourov, A. Tsygankov1, A. Rubin, M. 715–723 (2009). Seibert, Cyclic Photobiological Algal H2 Production, Sigma-Aldrich enzymes and reagents are (3) P. P. Peralta-Yahya, J. D. Keasling, Advanced biofuel pro- Proceedings of the 2002 U. S. DOE Hydrogen Program duction in microbes, Biotechnol. J., 5, 147–162 (2010). involved in many of these areas of research. Review (4) J. M. Clomburg, R. Gonzalez, Biofuel production in (9) A. A. Karyakin, S. V. Morozov, E. E. Karyakina, N. A. Zorin As summarized above and illustrated Escherichia coli: the role of metabolic engineering V. V. Perelygin, S. Cosnie, Hydrogenase Electrodes for Fuel and synthetic biology, Appl. Microbiol. Biotechnpl., 86, Cells, International Hydrogenases Conference 2004. 419–434 (2010).

Biomass O CH3

O HO

OCH CH3O O 3 OH

O HO HO OH OH CH3O O H O HO OCH3 HO OH CH3O HO O O OH HO O O OCH3

CH O OCH OH OCH 3 O OCH O 3 O CH 3 O 3 3 O Fuels

OH CH3 O HO CH3O Non-Renewable O O CH3 OH O O O OH O Fossil Fuels

OH HO

CH3 O H O OH H C H OH H HO OH Lignin Solar Energy O O Methane (Natural Gas)

CH2OH O OH OCH3 OH CH OH OH H H H O 2 HO O O H C C C H OH OH H H H CH2OH HO O O OH Propane OH CH2OH O O OH OH H H H H H H H

CH2OH O H C C C C C C C H OH O H H H H H H H OH CH2OH n O O Gasoline CO2 CH2O OH OH Cellulose O OH OH OH H H H H H H H H H H H H H H Fixation OH C Anaerobic H C C C C C C C C C C C C C C H OH H H H H H H H H H H H H H H Decomposition ombustion OH CH2OH CH2O CH2OH CH2OH O O O O Diesel Glucose OH OH OH OH O O O O O OH OH OH OH CO2 OH n OH OH Carbon Dioxide Starch OS O OH NH2 COOH S O CH3 S O O CO2 O OH H H3C CH2 CH2 CH2 CH2 CH2 CH2 C CH3 S O N CH2 CH2 CH2 CH2 CH2 CH2 CH2 O HO O OOH Carbon Dioxide

H3C CH2 CH2 CH2 CH2 CH2 CH2 C CH 2 CH C CH CH CH CH CH O 2 H2 2 2 2 2 2 CH2O OH O O OH H3C CH2 CH2 CH2 CH2 CH2 CH2 C CH3 CH C CH CH CH CH CH O 2 H2 2 2 2 2 2 OH HO H Coal OH Lipids OH Glucose

H2 Anabolic InVivo Enzymatic Conversions Hydrogen

H3C–CH2 OH Ethanol Microbial, Enzymatic and Chemical Conversions O H3C CH2 CH2 CH2 CH2 CH2 CH2 C CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 O BioDiesel

Renewable Fuels

Todays fuel sources originate primarily from the biosythesis of hydrocarbons originating from photosynthetic carbon dioxide fixation. The combustion of these fuels results in

production of carbon dioxide. Ideally, this might someday be a “closed-CO2-loop” system with solar power as the only energy input. Many of the structures shown are basic examples of simple unbranched molecules intended to represent heterogeneous mixtures. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 5

The New Enzyme Explorer Redesigned Format and Expanded Resources.

The Enzyme Explorer Indices provide The Enzyme Explorer Learning Center Allow the New Protease Finder to help paths to find more than 3,000: includes: you locate both the endo and exoproteases needed for precision protein cleavage. • Enzyme/proteins • IUBMB Metabolic Pathways • Inhibitors • Proteolytic Enzyme Resources • Substrates • Enzyme Assay Library • Cofactors • Carbohydrate Analysis Enzyme Resource The Enzyme Explorer Product Features • Product Guides and Protocols include enzyme and protein related The New Protease Finder • Request Enzyme Literature products for Cell Biology, Diagnostics and • industrial scale manufacturing. • Proteolytic Enzymes • Protease Inhibition • Diagnostic and Metabolic Research • Detection Substrates • Metabolomics and Dietary Research • Cell Dissociation and Lysis • Plasma Proteins • Carbohydrate Analysis • Proteomics and Protein Expression

sigma-aldrich.com/enzymeexplorer I I I I I I I I I I T L T E S P E S P Y E S E S S A C A C H A A R N A C D C A U R N R D N A N N A C D O O O O M M M M 6 M 2 P NH P P R P 2 CH P R R P CH R CH CH CH 2 1.2.1.32 CH CH D 3 N COO NH N CH N 2 N N N N N + N N N CDP 22 2-Amino NH 3 COO C N 22 NH 1.3.1.13 muconate C C C H 2.4.2.19 2 4.2.1.19 22 C C + C C C C 2.5.1.6 H N 1.2.1.32 (NH ) 2.7.4.6 OCH C H COO COO N O C OC COO N O C O C N HN O C C N NHCH CH SH 1.17.4.1 4.2.1.51 NHCH CH SH C (CTP) OOC NHCHCH SH NHCH CH SH COO COO N PPP HN 2

CH CH

2 HN R P

2 4.1.1.20 222 Quinolinate OC

R HC HN OOC 5.1.1.7 HCO H OH 22 Menaquinone 22 2 HN 2 HN C N NH + N 22 CYTIDINE- 1.5.1.7 - 10 2.7.4.14 3 (GMP) (XMP) 6-semialdehyde 22 CH COCOO CH SH 22 H CC OH triphosphate + + Degradation (IMP) Biosynthesis Degradation 22 N Biosynthesis + imidazole- 2-Aminomuconate- NH OC 22 CH COCH O P 22 pimelate 2 Diamino- - OH 4.3.1.3 3.5.4.10 Degradation Formylamido- COO 2 P nucleotide 4 2 6.3.5.2 6.3.4.1 Degradation 2222 6.3.2.1 Proton Flow 6.3.2.5 4.1.1.36 2.7.7.3 2.7.1.24 1.1.1.169 3.5.1.22 2.7.1.33 Plant Pigments INOSINE- P Quinolinate- Purines & Pyrimidines (SAM) C NH Amino Acids carboxamide-R OOCCH-CH CH CH CHCOO P GUANOSINE- Saccharopine OCH CH COCOO 6.3.4.2 1.1.1.205 XANTHOSINE- P P Imidazole Prephenate Phenylpyruvate 22 3 P P glycerol- NH CHCH CH COO COO C H OOC 22 3 1.14.12.1 R Coumarate SO acetol- P 3.5.1.18 CH CH 3 methionine S-Adenosyl Imidazole N N 2 32 HC Cinnamate CH(OH)CO + CH CH CH CH CH 2 SCH CH CH(NH )COO N N Adenosyl N N + SCH CH CH(NH )COO 4.1.1.45 Pantoate ) 1.14.13.11 )

2 32 erence Numbers of Enzymes of Numbers erence 1.5.1.9 322 CH=CHCOO 32 3 NH METHIONINE COO 3 OH C C Coenzyme A CH=CHCOO 2 Oxopantoate CH CH CH(NH )COO + Catechol

2.4.2.1 C CHNH CHCOO 2.4.6.7) refer to the IUBMBEnzyme the to refer 2.4.6.7) 5.4.99.5 drives the translocation of protons HOCH C(CH ) COCOO CH 2.7.7.4 LEGEND CH 3.5.4.12 O C 4- P -Pantetheine C NH NH COO 2 2 2 Urocanate HOCH C(CH ) CH(OH)COO PANTOTHENATE 4- P -Pantothenate 2.7.4.8 CH 2 32 (UTP) 2 32 2.6.1.5 HN 2.1.2.3 3 P Tannins LYSINE 2.6.1.9 N Glutathione

4.3.1.5 RP

3.1.3.5 CH CH 2.1.1.10 2.1.1.20 2 CH PPP CH N D CH CH OC + HN 4.1.3.27 Cytosine 2 32 R C(CH ) CH(OH)CO 2.4.2.19 NHCH CH COO NHCH CH COO 2 32 (NH ) O C URIDINE- N N OCH C(CH 24 N Anthranilate 4- P -Pantothenylcysteine CH Cysteine 6.3.4.4 Dephospho-Coenzyme A 2 32 22 3 C OOCCH(NH )CH CH CONHCHCONHCH COO N O 2.4.2.1 2 32 22 P H N(CH ) COMPARTMENTATION HOCH C(CH ) CH(OH)CO 3.1.4.6 ß-Alanine + 222 GLYCOLYSIS in the CYTOPLASM, GLYCOLYSIS 2 P and ATP FORMATION spanning the FORMATION and ATP triphosphate 2 NHCH CH CO Electron Flow P C C OCH C(CH ) CH(OH)CO carboxamide-R NHCH CH CO 2.1.1.13 2.1.1.14 HN OC CH CH NHCOCH The "Backbone" of metabolism involves The "Backbone" Glycine OCH 5-Aminoimidazole MITOCHONDRIAL INNER MEMBRANE MITOCHONDRIAL 3 d-UMP d-CMP d-CDP NHCH CH CO 2 OC NHCH CH CO OCH C(CH ) CH(OH)CO HN NH COO 2 OCH C(CH ) CH(OH)CO N + Pentose Phosphate Pathway Pentose from the matrix to intermembrane space. 2 32 2 P needed to form ATP from ADP and phosphate ADP from ATP needed to form 222 Biosynthesis NH Guanine P Aspartate CH O CONH Degradation NH Biosynthesis 3 Biosynthesis Degradation Photosynthesis Dark Reactions Biosynthesis NH retrolocation of these protons through the F0 subunits CH 2.4.2.18 N-Succinyl-2, 6 N-Succinyl-2, ADP- COO -ADP- CH CH(NH )CH O P

GDP Commission (EC) Ref (EC) Commission OOCCH CH CONH HC CH SH the TCA CYCLE (mainly) in the Mitochondrial matrix CH CH NH An electron flow (an electric current) generated from

NADH and UQH 4.2.1.49

+ NICOTINATE diaminopimelate OC-COO 2.4.2.11 P COO Vitamins Co-enzymes & Hormones 3 OH of synthase to the matrix then supplies energy ATP P + 222 (MELATONIN)

2.7.4.6 Small Numbers ( eg. ( Numbers Small 2 OOCCHCH CH CH CH-COO 1.3.1.13 RP The LIGNIN Human Metabolism is identified as far possible by black arrows black by possible Human Metabolism is identified as far 4.1.2.12 Carbohydrates 4.3.2.2 C NH + Lipids (APS) Tyramine (NH ) H CH 2.4.2.1 semialdehyde OCH P COO 1.2.1.25 OH UDP OH 2.1.1.45 2-Aminoadipate C 2 6.3.2.3 Chorismate 3 NH 2.6.1.17 CHCH(NH )COO 3 N 3 3 3 2 22 3 OHCCH CH CH CH N 3 O HC + CH HH OH C C CH CO CH C C Histidinol- P

3.5.4.3 CH + S-Adenosyl CH O 3 CH CH P COO Inosine 1.2.1.25 P P COO SCH CH CH(NH )COO Adenosyl 2.7.4.4 NH 22 R homocysteine C N 1.17.4.1 23 D D 2.4.2.4 Adenylylsulphate C-CH CH CH(NH )COO + 1.17.4.1 3.5.4.1 CH CH H OH 3 2.4.2.9 CH(NH )COO 1.2.1.31 322 Adenylo- VALINE Ribose- 1.2.1.25 3 succinate 2-Amino-3-carboxy + N-Acetyl-5-O-methyl-serotonin N N CH CH(NH )COO N O C N N COO CH + 3 HC + carboxamide-R 4.6.1.4 (NH ) CC H OH CH CHCO-SCoA 3 3 OOC-CH-CH COO 5-aminoimidazole- CHCH CH COO NH NH COO nucleotide 22 C 2 Nicotinate- C muconate semialdehyde muconate P CH LEUCINE PHENYLALANINE 32 I I + CH CH anthranilate OC HN 2

P -Ribulosylformimino

CoA R CH CH 2 3.2.2.2 ISOLEUCINE 2 222 CH (NH ) 3.1.3.15 CH

4.1.1.25 22 3 Isobutyryl-CoA 2 (UMP) 4.3.2.2 CH CH ++ N-(5-P-Ribosyl) pimelate 2.7.1.25 OCH 2 + N γ -Glutamylcysteine HCHO 32 2 O C N CH 2.1.1.4 propionate O CH CH Imidazolone OH 1.4.1.8 Bile Acids CHCOO HN OC P 3.3.1.1 32 2 CH N-Succinyl- 2.6.1.32 32 33 23 6 OOCCH(NH )CH CH CONHCHCOO Uridine- P O-C-COO OOCCH CH CONH HNCO I I (CH ) CHCH HSCH CH CH(NH )COO HN N 2 32 2.7.4.6 2-amino-6-oxo- 5.3.1.16 OC CH N -Trimethyllysine CH CH CHCOSCoA 1.13.11.6 THYMIDINE- P (CH ) CHCH COSCoA

2.7.4.9 + (CH ) N(CH ) Isovaleryl-CoA 2 Methylbutyryl- d-GDP CH CH(NH )CH OH 2.7.7.18 1.3.99.3 NH P OH OH COO N Fumarate 2 2.6.1.32 OOCCOCH CH CH CH-COO THYROXINE 2.6.1.6 1.4.1.9 OHCCOO HC 4.1.1.23 R 4.1.1.48 OOC-CH-CH COO P CH 222 1.14.16.1 HOCH 23 C C C NH -Adenosine P Glyoxylate 22 3 2 N H N 3 O O O O C N COO + 5-Amino-4-imidazole CHCOCOO C Ubiquinone Adenosyl 2 2-Aminoadipate COO Shikimate-5 3 2-Oxo- CH CH NHCOCH NH TDP 3 3 3 22 2 1.3.99.3 P 2 C C CH H 3.5.2.7 CH CH O N Adenine 1.3.99.10 Histidinol O O HC 2.4.2.15 HC 1.14.11.8 HN 4.4.1.8 4.2.1.22 CH O P CH + Taurine N isovalerate CH -SCH CH CHNH 3 222 NH

6.3.2.2 OOCCH CH CH CH Glutamate enolpyruvate 3- P enolpyruvate 2 + 3 CHCOCOO (NH ) 2 O C N OH pyrroline- 2.4.2.4 R CH glutamate C-COO NH OC 4-Hydroxy- 3 Methyl 4-Hydroxy- 3-Hydroxy- thiopropylamine (N-succinylcarboxamide)-R 2.6.1.23 4.1.3.16 NH 1.5.1.12 (Decarboxylated SAM) (Decarboxylated HO SCH CH NH NH CH CH-COO CH S-Adenosylmethyl CH = CCOSCoA HC O C N 5-carboxylate 2-oxoglutarate 32 Uracil 6.3.2.6 3-Hydroxy 2 2 CH OC HN O OOCCH(OH)CH COCOO 2.6.1.39 O 2.7.4.6 3 anthranilate acrylyl-CoA H C N 1.1.3.22 4.2.1.9 (PAPS) CH CH OOCCH(OH)CH CH(NH )COO 2.5.1.19 CH CH OH HN OC CH COCOO valerate 2 Pyruvate 22 3 3 sulphate - O - P - O - P - O CH 32 2 1.1.1.23 HC H C + 3 N + 23 2 1.5.1.2 P HO 23 TTP 2 Oxoleucine CH CH CH(NH )COO GTP TYROSINE MELANIN NH CH CH=CHCOSCoA 4.2.1.17 H 2.3.1.46 N Tiglyl-CoA 22 N-Acetyl-serotonin Piperideine- OOCC (CH ) CHCH COCOO d-CTP 1.3.1.2 d-GTP 2 CH 2-Oxo-3-methyl O P 23 2 H C OH 4.1.1.29 (CH ) NH CH CH(NH )COO 3 CHCOO O 1.8.1.3 3.7.1.3 CH 23 OH PEP CH 3-Methyl- C C R carboxamide-R 2.5.1.22 2.5.1.16 2 + 2 (AMP) O + COO CH CHCOO 5-aminoimidazole- CH Phosphoadenylyl- glutamate H H COO- 3 2, 6-dicarboxylate 2, 2 Desamino-NAD CH C = CHCOSCoA Formimino COO 2 NH CH CH(NH )CHO P -Ribosylformimino (CH ) NH COO 2 OH Ribose 2 H OH O C 2.7.7.6 N NH HOC-CH(OH)CH(OH)CH CHCH CH COO crotonyl-CoA CH 3 CH N CH C-COO N 22 3 N N H + 1.14.16.2 24 CH CHCOO Thymine CH 23 CH 2.3.1.5 + CH 4.2.1.17 CH 6 23 2 CH C + NH C NH 3 O O C (OH)CH(OH)COO 4.2.1.9 3 + H CC 1.1.1.85 N ++ N C CH C C 1.3.1.26 Xanthine Hypoxanthine HN OC (NH ) 3-OH-lysine C NH P(P) 222 3 2.6.1.5 COO OH Cystathionine Homocysteine 3 N -Trimethyl- CH CH(NH )COO SCH CH CH(NH )COO 2 (CH ) NH ADENOSINE- P H 2 CH CH(NH )COO R 2 3 CH O C CH NH HN 33 23 CH 2 Cysteate C HC OH OC CH OC HN HN CH CH-COO 6.4.1.4 2 OrotateP Orotidine- Succinylhomoserine CH CH 2 HOCH 2 isovalerate 2 6.3.5.1 6.3.1.5 CO NH Spermine OOC NH 2 N 2-Oxoadipate + 1.14.18.1 N 1.8.99.2 PROLINE Putrescine C H carboxylate Pyrroline-5- N 1.3.1.2 2.7.7.7 2 PROLINE 2.7.7.7 22 3

2.7.7.7 Spermidine (CH ) N(CH ) O HN OC C(OH)CH(OH)COO HYDROXY HN 2-3-Dihydroxy 3.5.4.19 OOCCH uracil CH OH CO P 1-deoxyribulose-5- HC CH CH 2 22222 2 24 OCH 1.5.1.2 OOC 3 323 Shikimate-3- P P 222 Histidinal 2 H NCH CH CH CH NH H N(CH ) NH 3-Hydroxy- C C 1.1.3.22

OH 2.7.7.6 P Dihydro- OOCCH CH CH COCOO CH CH-COO 1.5.99.8 4.2.99.9 Dopa 1.1.1.204 - HOCH CHCOS-CoA- 32 1.14.11.2 2.7.7.6 Homoserine 1.3.1.14 2.4.2.10 CH 3 3 HO SCH CH(NH )COO CH CH NH 3 C N NH carboxylate-R kynurenine 4.4.1.1 CO H C N HOCH CH CH(NH )COO Isobutyryl-CoA 1-(o-Carboxy phenylamino) 1-(o-Carboxy N COOH 2 23 NH 2.7.4.3 2.7.4.4 1.1.1.3 CH CH H hydroxy- H N(CH ) NH P CH CH(OH)CHCOSCoA 1.14.11.1 CH N 2 NH N malate 5-Amino-4-imidazole HC C NH COO 2-Methyl-3- O HC O 2:3-Di-OH- butyryl-CoA 2 2 2 222 3 2 3 HSO CH 3.5.2.2 1.1.1.23 C C CH-CH + dipicolinate 32 2.7.1.39 1.1.1.86 2, 3-Dihydro- 2, 2.7.1.71 3-Isopropyl- 2 + OOCC CH-COO CH 3.1.2.4 CH HO SCH CH NH O C NH C O N H HN 2 CH 3-methylvalerate H (NH ) Hypotaurine 4.1.1.28 Dihydro thymine (CH ) CHCHCH(OH)COO CH COCOO OH pyruvate N Creatinine ADP 1.14.13.9 C N O orotate 1.14.18.1 P 23 COO Dihydro P(PP) N 4.1.1.21 3-Methyl- OC HN (SEROTONIN) 1.1.1.35 OH HN OC R P 3 CH + COO OH DNA RNA Adenosine(P) CH CH(NH )COO + + 2 HO R OC HN + 1.17.4.1 4.2.1.33 O Hydroxyphenyl 3 3 Dopaquinone CH N N 222 4.2.1.52 (NH ) 22 4.1.1.17 COO OOCCH C = CHCOSCoA 1.1.1.86 NH 1.8.99.1 2 glutaconyl-CoA OH N OH COO C NH + 3.5.2.2 23 N Glutamic N CH 2 (NH ) 3 2 23 1.7.3.3 H C C 2 O O 2.7.4.6 N CH 5-Hydroxytryptamine Carnitine OH C 4.1.1.29 + COO COO P -Ribosyl-AMP CH CH(NH )COO 6.3.4.5 2.6.1.13 semialdehyde 2 222 1.3.1.13 C HC CH CO

NH C N

3 HO 2.7.7.7 22 3 CYSTEINE 3 HC ß-alanine N Glutaryl-CoA 2.7.7.6 NH + 33 2 NH OHCCH CH CH 22 1.1.1.3 Shikimate + HC C-CH(OH)CH(OH)CH O CH CO OH Carbamoyl NH CH CH NH N + 33 COO HSCH CH(NH )COO 1.2.1.41 OOCCH CH CH COSCoA O O PO HOCH CHCOO 3 H O HC (NH ) HISTIDINE 2 CH O 5-Amino homoserine 3-Hydroxy- O-Phospho- HS isobutyrate 4.3.1.3 OCH CH CH(NH )COO C H 3.5.2.3 COOH N (CH ) NCH CH(OH)CH COO CH COC(OH)CH HN Arginino- succinate 2 22 P Glutamyl- P CH COCHCOSCoA OC 2222 3 23 32 Kynurenine 3-Hydroxy

H NCONHCH CH COO 4.2.1.18 -O - P - O - P - O- N H 2 COO 1.13.11.20 imidazole-R (Vitamin E) 3.6.1.31 O PO O 2 2 (GABA) acetyl-CoA d-ATP d-ADP malate Dopamine 1.13.11.27 CH COC(OH)CH CH 2-Acetolactate + Aspartyl 3 butyrate 4.1.1.28 Cysteine hydroxy- 2 P OOCCH CH CH 32 ATP 2 O 223 sulphinate 2-Aceto-2- α -Tocopherol OC 4.2.99.8 2-Methylaceto- + ATP HN 1.1.1.25 Plastoquinone

4.2.1.18 ~~ 2.7.4.6 ß-Ureido (NH ) 4.2.99.2 H C

COO 2-Isopropyl- NH 1.6.4.1 3.5.1.6 3.5.2.10 2.6.1.19 4.1.1.15 Semialdehyde (CH ) CHC(OH)CH O P O HO SCH CH(NH )COO 1.3.99.7 4.1.1.70 OOCCH CH CH NH 3.5.1.9 23 P ) CONH OHCCH CH N P isobutyrate + 3 P Indole-3-glycerol- P -Creatine 222 NO Ribose + 22 6.3.3.1 O HNCN(CH )CH COO H C OH 4-Aminobutyrate H NCONHCH CHCOO 2 222 - CH CH(NH )COO 2.1.1.13 + + N O 4.1.1.22 (NH ) H NCNHCH CH CH CH OH P- 2 + 2.7.2.11 OOCCHCH COO H OH P + 3.5.1.6 2.1.3.3 + NH 1.14.13.39 R 222 3.5.2.5 2 4.2.1.20 CH COO

CH-COO 1.2.1.11 OH

CHO

1.1.1.31 4.6.1.1 NHCOCH CH NH H CC 1.14.17.1 4.3.2.1 2 CH CH NH N H 2 2 + CH COO 2 3 3.5.1.2 6.3.1.2 Carnosine + 23 NAD( NH + NH 2.7.3.2 2 aspartate O COO

OOC CO 22 CH CHCOO 4.4.1.15 NH 5-Hydroxy- Dehydro- - Carbamoyl NH shikimate tryptophan 3 OC OH OH HISTAMINE C NH C NH 32 C NH CITRULLINE OCH 23 2 CH 23 23

H + 4.2.1.20 OCH 3 N HC P -Ribosyl-ATP H C CO CH CH(NH )COO Formyl CHO C (NH ) HO CH CH COCOO COO 3.5.3.6 P 22 N CH CH(NH )COO COO OH 223 33 22 6.4.1.3 CHOHCH OH CYSTINE OH Succinic 2 22 pyruvate HN CH CHCOO 3 4.2.1.16 N 2 22 C H COO Glutamine Oxobutyrate 3 N 1.13.11.11 4.1.3.18 2 222 2 ß-Alanine H NCH CH COO S-CH CH(NH )COO S-CH CH(NH )COO

4.1.3.18 OOCCH CH CHO HC 3-Sulphinyl HO SCH COCOO COO Acetylserine HC 22 6.3.5.5 CH CO-OCH CH(NH )COO THREONINE 2 H NOCCH CH CH CH CH(OH)CH(NH )COO NH O 4.2.1.10 UREA H NCONHCH CH CH CH + H NCONH N H + NH 3 (NH ) Homogentisate 2 semialdehyde 2 NH OH (NH ) N 3 - OH

3 3-Amino-

22 + glycinamidine-R 2.6.1.22 - 6.3.5.5 Aspartyl- P 2 N NH (NH ) CH OC 2 COO isobutyrate CH 3 P H NCH Allantoate Allantoin URATE OH CHOHCH NH 1.14.16.4 + HC HN OOCCH CH 32 2 32 Formylkynurenine OH phenylglycol Creatine 3.5.3.1 CH 4.1.2.5 C1 P COO H NCN(CH )CH COO OHCCHCOO P CO ATP 6.3.5.3 22 HS 2 OH

2.3.1.30 CH CH COSCoA 2.1.3.1 P HSO ACID HO 1.4.1.14 4.1.1.41 5.1.99.1 4-OH-3-Methoxy- POOL

Cyclic AMP Cyclic quinate FOLIC 1.13.11.5

+ 4 2 (Noradrenaline) 2.1.1.6 O PO NH O 2.1.3.3 O Dehydro- Propanoyl-CoA Methylmalonyl-CoA 2.7.2.4 NH semialdehyde Methylmalonyl OOC-CH-COSCoA 3.5.3.4 CH COO P Norepinephrine semi- 2.6.1.18 O TRYPTOPHAN 2.3.1.16 R 2.1.1.2 4.1.1.11 O Malonic 4.2.1.22 OHCCH 2 2 NH ARGININE aldehyde OOCCH CH CH GLUTAMATE 22 COO CHO H NCOO 3 Indole Indoxyl 3 + ORNITHINE O NH COO NH NH 4.6.1.3 2 222 22 Urea R-CO-COO + 2.1.3.2 2 222 6.3.4.16 Maleyl H NCH CH CH CH OCH Carbamoyl- (NH H NCONH 4.1.2.5 H NCNHCH CH CH CH COO 1.4.4.2

4.1.99.1 1.4.1.19 2.1.4.1 2 2-OXO ACID 2-OXO 2.1.1.28 Formyl 4.1.1.28 OC 3 OH HC

CHOHCH NH

acetoacetate 1.6.6.4 6.3.5.4

2 1.18.6.1 + O 1.7.7.1 1.4.1.2 2 I + 222 2 2

2 T 2.1.2.1 CH CHO - 5.2.1.2 CH COO glycinamide-R 2 - A Glycine 2 (Normetadrenaline) 2 N Asparagine CH CH NH 3.1.3.3 23 2.6.1.- R-CH(NH ) CH COCOO NH

22 1.2.1.16 N 2.1.2.2 OH 4.1.1.12 1.2.1.18 I 3 23 22 CH COO + CH NH COO NH + Normetepinephrine O H NOCCH CH

4.3.1.1 M 22 ASPARTATE SERINE 4.1.1.71 2-AMINO ACID + 1.2.1.4 (Auxin)

NH 2 5.1.99.1 5.4.99.2

NH A NO H NCNHCH COO C OH 1.4.3.4 + 33 Indole-

3 S 2.6.1.52 N NO 23 OC 33 1.5.99.2 2.1.1.5 A O Guanidoacetate HOCH CH(NH )COO 2

2 R 3 LACTATE T 1.2.3.7 CH CH(OH)COO H OCH Tryptamine OH ALANINE CHOHCH NHCH serine NHCOCH NH 4.1.1.43 + 3 HOCH HCOH Indoleacetate P 1.6.6.1 1.7.99.4 CH CH(NH )COO + 3 CH (NH )COOH N(CH ) + 2.4.2.17 acetaldehyde 23 32

2 33 2 32 GLYCINE (Adrenaline)

Indolepyruvate 2 33 OCH CH(NH )COO OCH COCOO Phospho- OH 2.1.1.20 4.2.1.52 OCH CH CHO 2.6.1.51 1.2.1.8 1.1.99.1 P CH COO P pyruvate Epinephrine 1.4.1.7 P CH CH OH 2 22 O Fumaryl 1.1.1.1 P -Hydroxy- OH ACETATE CH(OH)COO 2 -OOC heptulosonate-7- P ETHANOL D-mandelate OH Betaine Betaine 3-Deoxy-D-arabino-

2.4.1.22

aldehyde

acetoacetate CHOLINE 1.4.1.10 1.1.1.29

pyruvate

Sarcosine 4.1.3.18 OOCCH HOCH CH N(CH ) 2.6.1.4 Hydroxy- Acetaldehyde CH O OOCCH NHCH OHCCH N(CH ) 4-OH-3-Methoxy- OOCCH N(CH ) 2.6.1.2 1.1.1.27 HOCH COCOO ribosyl- P - 1 Glycinamide- Dimethylglycine - - F P 2.6.1.1

β OH

0 O OPPU P COO COO - e Glycine

s F 4.2.1.3 OH OH 2 2 + O

a T

CO.SCoA H 2 α - O

OH h s

6.3.4.13 3 3

2.3.1.37 t 2 t +

A CH C(OH)COO CH i Glyoxylate n 1

H COO

1.4.1.1 2 CH OH y n OH - 2 OH CH OH

s F CH OH 4.4.1.15 - u 2 α

OH XO- 1.1.1.41 - CH OH

P ε

3.2.1.23 O CH 2.7.1.38 +

1.2.4.2 2.3.1.61 b OH c 4.1.1.1 4

2 T NH

H 4.1.3.7 HO HO O P COO u 3

4.1.1.32 A . 2

2 s 5-Amino- levulinate HO

1 CITRATE

O - +

COO . γ

OH g γ

OH c

ATP OH 2-O 6

CH δ +

CH(OH)COO CHCOO CH C

- . n ADP +

OOCCH i i P

6.3.4.7

O - + + 3 t 0 1.1.1.95 ISOCITRATE 6.2.1.4 ATP H 2.4.2.14

2 r OH GLUTARATE P 1 2H 2.7.1.6

H H OH HO 2H o 2 OH

GTP amine

2.7.7.10 2 1 COO p 2 + + oscp P

O 4.1.3.8 GDP

+ + OH s 2 H

OOCCOCH β + 2.7.1.2 2.7.1.1

CH O CO O ATP - OH n 2

OH P -Ribosyl SUCCINYL-CoA ADP

CH OH CH OH 2 2 2 α CH OH

H H

LACTOSE a

GALACTOSE

OPPU

HOH β 2 r CH

ADP P Galactose- P NAD+ t 5.1.3.2

CoA - 2 a OH

ATP β CH OH CH O 4.1.3.1

O + Glycerate CH(OH) -

Pi HO UDP -Galactose D + OH CH OH

H A

CO NADH P N HO + - 2 HOCH 2.7.1.11 _ OH + GLUCOSE HO

P P

2.7.7.12 6 O H CO +

H - OOCCHO - I OH Glyoxylate Cycle 1.8.1.4 H 1.2.4.1 CH OH 2.7.1.31 COO F HO 2.3.1.12 O COO COSCoA COO OOCCH

H + T - 3 P 3.1.3.9 δ O H 1.2.1.12 HO

H C HO + P H COCOO HO CH UQ . 2

1e-

3 2 A i - H SUCCINATE H CHOHCOO H

OH 2 E Pi P 2 4.1.3.2 5.4.2.2 2 H CCC OH OH 2.7.7.9 CH OH 2 CH 1.2.4.1 2.3.1.12 3.1.3.43 2.4.1.11 2.4.1.21 1e- ATP

UQ R 2 CO.SCoA COO 2 H CCC OH OH 2 5.3.1.9 2 HO 3.1.3.11 4.1.2.13 + OCH OCH OCH CHOH

ACETYL C 5.4.2.1 2.7.1.40 PYRUVATE CH =C(O ) Pi CH(O ) CHOHCHO UQ H 1:6-bis- P I UDP -Glucose P P P 2 Fructose- HOCH 4.2.1.2 4.2.1.11 Cyt.b OCH Glucose-6- P 1.3.5.1

N OOCCH=CHCOO + 2

FAD ADP + ADP ADP - P etc. PYRUVATE 2.7.2.3

or O NAD H 2- P -Glycerate 3- P -Glycerate 2.4.1.1 OCH P Glucose-1- P 1.1.1.37 + + + ADP + P

1.2.1.13 G P -enolpyruvate 2H ATP 2H CH(OH)CH Fructose-6- P 2 III OOCCOCH

3 R - OXALOACETATE 2H COO L FUMARATE 6.4.1.1 ) 4H 2H 2H 1:3-bis- P -Glycerate CH O

II E MALATE 3- P -Glyceraldehyde FAD CH P ADP 2.7.7.27 4.1.1.9 ATP D 3 1.1.1.39 Fe-S 2

+ N CO 2.4.1.21 ADP - .

_ + 1.1.1.49 2 5.3.1.8

Cyt.b CH(O

2 2.6.1.16 5.3.1.1 2 E 2e- + 2 IV Cyt.b 2.7.1.28 Glucose P

A H O I + 2.7.7.34 NH OH 2 2

2 2.6.1.44

2 XY

3.7.1.2 1.9.3.1 glycerate CO O 5.3.1.1 ATP OCH PP 1 NAD H 2UQ

Cu NADP 3.2.1.48 CHO 2UQ 2 OH 2.2.1.2

CH O 1.1.1.21 PP 2 UQ H 2.2.1.1 2.4.1.29 NAD UQ H

A Heme a OO H 2 H 2, 3-Diphospho- 2, 1.6. 5.3 OH H C OPPT O UQ H

B FMN H OPPT

HO OH 2Fe -S -S 2Fe 1.10.2.2 2e-

HOOCCH CO-SCoA 2.7.7.24 2.6.1.4 OH 3 O Malonyl-Co-A Heme a NADH+H Cyt.c Fe-S

Cu PROTONS 2e- OH OH OH O H CC 2 H C CC OH OH I 1.10.2.2 OH 2.3.1.38 / P O O 3 (5 Clusters)

CH CO-S-ACP

H+H NAD 1 Glucosamine-6- P 2.2.1.1 GDP-Glucose P FMN O 1.6.5.3 2.2.1.1 OH 2 3 D OH

2.3.1.39

UQ 4.1.3.4

2 1.1.1.22 4.2.1.18 3.2.1.26 2 CH OH 2 2 OH E lactone CH CH O COO Acetyl-ACP T 4.1.2.- + O P 2.4.1.13 OH OCH + + P -Glucono CH O

2 A Cyt.c OCH P P-Ribosyl- Sedoheptulose- PP O HO

Glyceraldehyde C P P Erythrose-4- LO HO TRANS 2H 4H 4H TDP -4-Oxo- HOOCCH CO-S-ACP NADPH COCH Malonyl-ACP

2.3.1.4 2 4.2.1.46 2

4.1.3.5 TDP -Glucose NADPH TDP -Rhamnose CH OH 6-deoxyglucose 2.3.1.16 5.3.1.8

5.1.3.13 2.3.1.16 PP OH O-ANTIGENS STARCH GLYCOGEN 2.7.6.1 2 2.7.1.4 P ) (Glycerone- HOCH PP OH 2.3.1.41 PP 2.3.1.41 2.3.1.41 CO 1.1.1.30 acetone- P 2.4.1.9 Dihydroxy- P CH 4.2.1.24 2.3.1.41 O O INULIN CELLULOSE PP

OH 3.1.1.17 OH C CHO H 2 2 2 OH 2.3.1.9 O + SUCROSE OHHO OH C=O H 32 CH CH O CH O - COO- 2 2 2 2 5-Amino- levulinate 5.5.1.4 2 - C 3 2 (C30) H CC OH H (C15) 2.7.1.7 2 4.1.3.5 2.7.4.2 2.5.1.1 2.5.1.21 2.7.1.36 2 5.1.3.1 1.1.1.32 2 (C10) 4.1.1.33 2 1.1.1.34 2.5.1.10 CH CH(OH)CH COO OH HO CH OH CH COO 2 CH CH CH H CC OH OH 2 2 HO (C5) 3-OH-Butyrate 2 COCH COSACP 32 CH COO COO COCH COSACP 22 CH COO Squalene COO NCH CH COO 32 CH OH 3 22 mevalonate Diphospho- NADP COCH COSACP 2 3 22 2 32 CH OH Mevaldate Thromboxane B2 Thromboxane CH HO N H 3 2 14 2 HO CH C(OH)CH CHO Mevalonate 3 22 5.1.3.1 H H OH PP Farnesyl- PP Geranyl- CH C= CHCH O D-Ribose-5- P GDP - CH COCH COO glutaryl-CoA MANNOSE CH C(OH)CH CH OH D-Xylulose-5- P OCH 1.1.1.132 OH CH C(OH)CH CH O CH C(OH)CH COSCoA C H COCH COSACP Mitochondrial CH (CH ) COCH COS-CoA P Oxostearoyl-CoA ß-OH-ß-Methyl- CH C-CH H O 5.4.2.8 Acetoacetate 3 3 OCH 2 3 2n 2 OH P 2 3 22 2 2 Mannose-6- P 2 3 26 OH C 2 PP Isopentenyl- ALGINATES HC CH H CH OH HC KETONE BODIES KETONE 4.1.1.4 HN Mannuronate CH (CH ) OOC - + 33 CH (CH ) 3 H OH 3-Oxoacyl-ACP CH OH 5.4.99.7 COO CH (CH ) 2.4.1.33 1.14.99.7 OH n OH H H C COH OH CO Oxalate CH COCH HOOC-COOH OPPG 3 CH 5.3.99.5 Galactose 32 Acetone P P H CCCC OH H 32 O NHCOCH + 6- P -Gluconate 2 2 2 33 3-Oxo-Hexanoyl-ACP 1.2.3.5 O (C20) OH H 2.3.1.6 3-Oxo-Decanoyl-ACP 1.1.1.44 2 PP H CC OPPG NH 2 2 CH COCH COSCoA P 2 32 2 - Sorbitol 2.7.7.13 CH C= CHCH 2 - HO 3 H OH Mannose-1- P 3 22 2 Galactose 32 2 O O 2 3.1.2.11 2 2 2 OH COO 2 2 CH O CH O 3 PP CHOLINE 2 CH CH COCH COSCoA 3 HO OCH 3-Oxoacyl-CoA OH CH 2 O 22 CH (CH ) COCH COSCoA CH (CH ) COCH COSCoA 4.3.1.8 HOCH CH N(CH ) 2 H OH 2 4.2.1.75 COO CH P 1.1.1.100 CH OCH COO 2 CH Acetoacetyl-CoA - CH 5.3.1.6 3-Oxohexanoyl-CoA NH (Vitamin E) + P OH 2 3 2 2 33 1.1.1.8 CH OH 2 HO 2 2 OHCCOO CH Acyl-CoA HOCH HO Glycol HO CH O 3-Oxopentanoyl-CoA (C5) 1.1.1.100 HOCH CHO H OH C C C CO 3 P D-Ribulose-5- 3 O OH HO HOCH COO 3 Acetylcholine 1.1.1.100 CH OH CH COCH CH N(CH )

1.1.1.100 3.1.4.2 α -Tocopherol Phytol NHCOR aldehyde COO CH HOCH CH Glycolate N-Ac-Glucosamine-6- P CH CH COO HO CO 2.5.1.29 2.7.1.3 1.2.1.21 1.1.1.14 Glyoxylate 2.7.8.5 1.1.1.79 2 CH 1.4.3.8 OH P CH H CH 32 N N Fructose 2.4.1.69 2.4.1.68 3 2.7.1.32 HO H H Ethanolamine Fixation Ribulose-1,5-bis-P OH 2 2 2 2 Endoplasmic Reticulum CH C = CHCH O H OH

2.7.1.15 (C20) GDP -Fucose C C H H 5.4.2.3 HO CH H + 4.2.1.47 HOCH - -- Psychosine CH OH CH O Leukotriene B4 Leukotriene GDP -Mannose P SUBSTANCES 3- P -Glycerol PP Dimethylallyl- H 2 H OH 3 2 2 C C C CO H 2 2 1.1.1.157 ATP + 1.1.1.35 1.1.1.35 1.1.1.35 2 BLOOD GROUP OPPU 2.7.8.5 + N N HOCH Fructose-1- P 2 Cerebroside 2 2 CH OH P CH CH 2 COO CH CH COO O OH O - P

P 2.7.1.82 H 2 2 33 3 2 14 2 NADP e HO O 2 OH 2 OH H HO O 2 2 3.1.4.4 HC β OH 1.10.2.1 1.10.3.3 2 2 CH (CH ) COCH COS-CoA O 2.7.1.30 HO NHCOCH 5.1.3.6 HOCH COO H PP Geranyl-geranyl- OH 3 2 12 HC HC UDP - OCH CH N(CH ) α 2.7.7.41 β PGE Prostaglandin CH O-CO-R CH O 2 Chain elongation 2 OOC OOC OH H - - PECTIN OH + 2 2 OH CH(OH)CH COSACP ADP O CH (CH ) CH=CHCH(OH)CHCH O- HO CH(OH)CH COSACP Choline- α 3 CH(OH)CH COSACP OH H C COH C CHO OH β 3 2 12 CH OH α 2.3.1.51 Phosphatidate 1 β 2 Ribitol 2

Inositol- P i HO 2.4.1.23 OH 3 H CCC OH OH w 5.1.3.4 32 2 α H D-Ribose 2.4.1.47 22 223 CH (CH ) CH=CHCH(OH)CHCH O-

Galacturonate o 1 P 2.7.1.17 HO ATP l CH OH 3.6.1.34 O α CH OH

i 2.5.1.32 R’-CO-OCH f + 1.13.11.34 ADP NH CH CH(OH)CH COS-ACP 2 2 3 2 14 3 22 H + 3-OH-Acyl-ACP 3 2n ATP OH CH CH N(CH ) 3 26 n 4.1.1.37 P - OPPU n 2.7.7.15 HOCH H + COO - 3 glycerol o + +

) 1.3.99.7 CH (CH ) CH(OH)CH COS-CoA CH (CH ) CH(OH)CH COS 2 2 - CH (CH ) O OH t O HOCH (C40) + CH (CH ) r synthase ATP 3 + - CH (CH )

O 5.3.99.3 O n n O

t H 3-OH-Hexanoyl-ACP H 22 (Vitamin K) O H 3-OH-Decanoyl-ACP Glycerol O OH C CH CO CO CO NADP H+H H CDP-diacyl

e H 2.7.1.47 H 2.7.1.107 NH +

HOCH c

r CH CH O-CO-R COO H 2 2 2 2 + O-PO 2 r 2.3.1.15 2 2 2 CH 3.1.3.25

1.14.99.1 2

UDP -Glucuronate e

UDP-Galactose

Phytoene 3

u 3 2 3 l H 2 2 CH CHOHCH OH 2 2 OPPU

COO c OH 3.1.4.3 3.2.1.46 OCH CH 32 2 CH OH CH OH CH OH Phylloquinone CH CH O POCMP CH OPO CH O-CO-R 5.1.3.12 OH CCCCCO H CH OH

e 2 CH

c CH CH CH(OH)CH COSCoA 3 22 3 P 3-OH-Acyl-CoA

ASCORBATE i UDP - CH + 8 O

γ 8 OH

r ε

ε CH CH COO 3.1.4.12 OH t

c HOCH CH CH CH(OH)CH COSCoA

i c Dehydroascorbate 2 O O

CH (CH ) CH(OH)CH COSCoA N CH (CH CH(OH)CH COSCoA

l N

3.1.3.4

4.2.1.60 3

(Coenzyme Q) HO e 3-OH-Butanoyl-CoA Iduronate HO HOCH

Ubiquinone Menaquinone Plastoquinone H 4.2.1.60 4.2.1.61 l

+ 4.2.1.60

Ethanolamine- P c 2 2 N-Ac-Glucosamine-1- P H + -CO-OCH 3-OH-Pentanoyl-CoA

' CH OH +

H e 4.2.1.59 OH 2 2 -CO-OCH 4.2.1.58 NHCOR

2 2 33

( y Glycerophosphocholine + H COO R 3 CH Phosphatidylglycerol

C C H H H

R H c + CH O-CO-R CH OH

OH a

H 3.5.1.23 OH - H H + - CH CH N(CH ) OH O OH O HOCH H -- +

CO-S-ACP OH OH

HO H Serine Inositol H n 2 2 2.7.7.23

2 2 H

CHO N N o c Arachidonate Inositol 3 3

COO OH CCCO HH 4-Sphingenin - OH C CH CO OH OPPU VISION H C COH C CHO OH

CH COO COO CH H CCCO OH H 2 HO N H 2 4.2.1.17 CH CH COO 4.2.1.55 4.2.1.17 3 +

CDP-choline 2.7.7.14 2.4.1.17 2.7.8.3 R’-CO-OCH CH O CH O C PP -O O 2 H NHCOCH 2 2 hv 2 2 Diacyl CH OH H CH CH=CHCO.S-ACP D-Xylose COO H C COH OH CO HC glycerol D-Xylulose 3 2 12 2 2 + R-CH 3

3 3.1.1.5 UDP -N-Ac- 2 HC OCH OH - HC H OCH Ceramide L-Xylulose-5- P l 2 1 CH (CH ) CH=CHCH(OH)CHCH OH P CH OH + + P HOCH 5.1.3.7 HOCH * A Galactosamine P700 H H Fe-S + 2.7.8.8 1.13.99.1 CH O-CO-R’ COO Chl.A 2.7.8.11 2e HO PHOTO- SYSTEM

n H FATTY ACID FATTY HCO-CO-R + HOCH 2.3.1.20 O 1.1.1.105 -

o + Desmosterol Zymosterol Lanosterol i 2.7.8.2 CO H CH=CHCOSACP To Brain - To t CH=CHCOS-CoA + 3 2 12 O OH O H CH=CHCH COSACP Ferredoxin 2 2 a Acyl-CoA 2 2 l PC 3 2 14 H 1.3.3.3 3 5.3.1.3 3 + y 2 3 + CH (CH ) CH=CHCH(OH)CHCH OH H r + + Glucosamine 2.7.1.53 CH OH CH OH 2 2.7.1.16 H 11- cis -Retinol 2.7.1.47 CH OH CH CH H CH (CH ) COSCoA o H CH

PC + - + HO Rhodopsin CH CH=CHCO-S-ACP 3 NH 2 2

h 11- cis -Retinal + COO OH Palmitoyl-CoA 3 2 14 n 3 2 2 3 26 2.4.1.16 3 22 3 2n + Crotonoyl-ACP 3-OH-Butanoyl-ACP Acetoacetyl-ACP CH 23 p H + 3 25 + OH C CH OH CO +

PC H H 2 2 33 NH OPPU s PQ O + H 2, 3-Enoyl-ACP 2, CH O-CO-R CH O-CO-R" OH C CHH C CO CO OH OH 3.1.1.32 OPPU H CH (CH ) CH=CHCOS-CoA H 3 H CH CH (CH ) CH CH COO Palmitoleoyl-ACP CH (CH ) CH=CHCO-S-ACP o CH (CH ) CHITIN CHONDROITIN 2 O CH (CH ) 3, 4-Decenoyl-ACP 3, 22 1.1.1.9 + NHAC - O -

2, 3-Hexenoyl-ACP 2, h 32 CH CH N(CH ) NHAC + Lysolecithin N 2, 3-Decenoyl-ACP 2, N FAT

3.1.4.12 Cyt.f OH H CH CH=CHCOSCoA 2 2 3.1.2.20

4.1.3.20 3 22

32 p -Linolenate O 1e + H H H O H H 2 OH OH OH HYALURONIC ACIDHYALURONIC DERMATAN 2PQ

- COO

- o Dark - 2 2 Light CH CH CH=CHCOSCoA

- γ

H Crotonoyl-CoA t 3-Enoyl-CoA 2, 5.2.1.7 Pentenoyl-CoA 222 Opsin OCH CH O 5.2.1.3 O R’-CO-OCH CH OH O CH (CH ) CH=CHCOSCoA CH (CH ) CH=CHCOSCoA H C C H

2, 3-Dioxogulonate 2, O o 2 2 33 2 2e Xylitol 2e + HOCH 2e + 2 CH OH OPPU H CCC OH OH

- 2 h + 2, 3-Hexenoyl-CoA2, 3-OH-Hexanoyl-CoA OH CCCO H OH 1.3.1.10 _ OH C CHH CO H C COH C CHO H . H - 1.3.1.9 H HO H C H

COO HOCH Triacylglycerol

3.1.1.3 2 P 2 2 O CH CH N(CH ) CH O-CO-R CH CHNH 2 - CH O PO H HO SERINE + N N -

Cyt bc 1.3.1.9 2H

- CH OPO 2e 2 Cyt bf 2 Glucuronate HO CH O-CO-R CPP- 2

c CHO

2 3 1.3.1.9

COO- UDP -N-Ac- PQ 1e 2 i LECITHIN

2.7.8.3 L-Xylulose

+ 1.3.1.9 + O O O l O CH 2 Cardiolipin CH CH CH COO CH OH

_ L-RibuloseP L-Ribulose-5- Fe-S

UDP-Sugars . HOCH CH c

NHAC H H D-Arabinose D-Ribulose

- - OH O-CO-R CH CH y 3 2 14

2 CH OH 2 2

-CO-OCH + 2 2 33 6.2.1.3 2 2 COO ' CHLOROPHYLL C + 2e COSCoA PHOSPHATIDYL HC HOCH CDP-Ethanolamine + 1.1.1.130 R HOCH COSCoA 2PQ HOCH CH (CH ) CH(OH)CHCH OH CH O-CO-R CH CH(OH)CH O-P-OCH H 3 CH O-PO CH OPO CH CH N(CH ) COO 3 HOCH HO CH H 2 2 STROMA 4H + pyruvate HC 1.1.1.158 1.14.99.25 HC - 1.3.99.2 1.1.1.10 1.3.99.3 1.3.99.3

5.1.3.14 2.4.1.62 - UDP -N-Ac- OH 6.3.2.13 PQ PO + O O + CHOLESTEROL 1.1.1.102 1.1.1.19 GLYCAN Protoporphyrinogen Coproporphyrinogen Uroporphyrinogen Porphobilinogen 6.3.2.7-10 PEPTIDO- Glucosamine 2H THYLAKOID LUMEN H H -CO-OCH 1.3.99.7 ' 1.1.3.8 PQ H (C40) O UDP -N-Ac-Muramate HO 2 2PQ H R THYLAKOID MEMBRANE COO 2.7.8.1 (C40) OH OH (Vitamin A) 2 R'-CO-OCH Metarhodopsin Protons from Water 1.1.1.105 2 4.1.1.34 3 Translocated protons Translocated trans -Retinal 2 + + CHLOROPLAST OUTER MEMBRANE CHLOROPLAST OH 2.3.1.7 1.3.1.35 5.3.1.4 trans -Retinol 2 CH OH 3 CH CH COSACP H OH H CH - CH CH COSACP CH H NHAcyl PQ H CH - 3 OH H 1.3.1.35 3 2 2 n+ + OH OH NH CH 3.1.3.29 4.1.3.20 HO 2.7.1.60 2.7.7.43 H H H 3.1.3.29 OH 1.3.3.4 2.1.1.71 2 - 1.13.11.21 4.99.1.1 + NH HH OH CCCCCO H HH OH C C CO C 2.1.1.17 COO CH CH 2.4.99.7 CH 3 2 14 + O COO 322 (Sialate) OH 4.1.1.65 OH - OH OH H 2 3 26 2 2 OH 3 22 2 2 N O COO N Oleoyl-CoA (Cytosol) - Linoleate OH 22 H OH H n H OH H OH OH 2 A CH (CH ) COS-ACP B OPC 32 O Decanoyl-ACP Hexanoyl-ACP Butanoyl-ACP ACYL-ACP Palmitoyl-ACP CO P CH CH CH COSACP Stearoyl-CoA Dehydrostearoyl-CoA OH-Stearoyl-CoA O + Lycopene CH (CH ) Q Q 322 CH (CH ) 2e 2 2 33 Mn 2e 2 PHOTO- SYSTEM II O 2 ACNH H C C H CH (CH ) COS-CoA MUCINS P680 Gulonate OH CCCC H HH Chl.a H - Fe O 3222 OH CCC HOH OH C CH C CHO OH OH C CH C CHO OH ACNH OCH CH ACYL-CoA OH C CHH C CHO - O HOCH CH CH CH COSCoA O - OH 3 22 2 2 CH CH N(CH ) + CH OH OH HOCH 3222 Butanoyl-CoA CH O Hexanoyl-CoA 2 3.1.1.28 O-Acyl-carnitine 2 2 2 OH O O 2 2 Pheophytin 2 CH CH CH CH COSCoA O-Acyl-carnitine Pentanoyl-CoA 3 2 14 N-Ac-Neuraminate 2 2 - H N N 2 (Mitochondria) 2 2 ACYL-CoA CH (CH ) CH CH COSCoA Odd C Fatty acids C Fatty Odd O O O CH (CH ) CH CH COSCoA 2 L-Lyxose CH CH CH COO HO CH Gulonolactone 2-Oxogulonolactone 2 HEME CH OH 3-Dehydrogulonate inositol CHOH CHOH HO L-Xylose CH (CH ) COCHCH OH Serine ß-CAROTENE 2 L-Arabitol CH CHOH CHOH CH O-CO-R HEMOGLOBIN CH O-PO 2 2 HO Choline 1.14.99.5 CEPHALIN L-Arabinose HO Carnitine CH O-CO-R CH O P HOCH Retinoate neuraminate STEROIDS HOCH 1.2.1.36 3.1.1.21 HOCH 2.3.1.76 HC HOCH HOCH CMP -N-Acetyl Phosphatidyl H N-Ac-Mannosamine Phosphatidyl CH OPO -CO-OCH ethanolamine CH OCH=CHR SPHINGOMYELIN ' 3 3 Progesterone 2.3.1.50 3 2 12 Dehydrosphinganin Sphinganin Retinol esters plasmalogen Pregnenolone R Gangliosides GANGLIOSIDES HC HC 3.1.1.18 AcNH 1.1.1.45 -CO-OCH AcNH CH (CH ) CH=CHCH(OH)CHCH O ' GLYCOPROTEINS R N-Ac-Mannosamine-6- P R-CO-OCH I I I I I I I I I I I I I I I T T T L T T L T L L P S Y E S S S P E S E X S E S P E S E S P S Y E S S E P S P P S S E S P P Y S P Y S E S P H N H R N D H N D B N H D R A D A N H H D R D R H R N A C C H A R D D O O O O O O O G O O O O O O Metabolic Origins of Fuel I I I I I I I I I I T L T E S P E S P Y E S E S S A C A C H A A R N A C D C A U R N R D N A N N A C D O O O O M M M M M 7 2 P NH P P R P 2 CH P R R P CH R CH CH CH 2 1.2.1.32 CH CH D 3 N COO NH N CH N 2 N N N N N + N N N CDP 22 2-Amino NH 3 COO C N 22 NH 1.3.1.13 muconate C C C H 2.4.2.19 2 4.2.1.19 22 C C + C C C C 2.5.1.6 H N 1.2.1.32 (NH ) 2.7.4.6 OCH C H COO COO N O C OC COO N O C O C N HN O C C N NHCH CH SH 1.17.4.1 4.2.1.51 NHCH CH SH C (CTP) OOC NHCHCH SH NHCH CH SH COO COO N PPP HN 2

CH CH

2 HN R P

2 4.1.1.20 222 Quinolinate OC

2 32 erence Numbers of Enzymes of Numbers erence 1.5.1.9 322 CH=CHCOO 32 3 NH METHIONINE COO 3 OH C C Coenzyme A CH=CHCOO 2 Oxopantoate CH CH CH(NH )COO + Catechol

4.3.1.5 RP

GDP Commission (EC) Ref (EC) Commission OOCCH CH CONH HC CH SH the TCA CYCLE (mainly) in the Mitochondrial matrix CH CH NH An electron flow (an electric current) generated from

NADH and UQH 4.2.1.49

+ NICOTINATE diaminopimelate OC-COO 2.4.2.11 P COO Vitamins Co-enzymes & Hormones 3 OH of synthase to the matrix then supplies energy ATP P + 222 (MELATONIN)

P -Ribulosylformimino

CoA R CH CH 2 3.2.2.2 ISOLEUCINE 2 222 CH (NH ) 3.1.3.15 CH

NH C N

4.2.1.18 ~~ 2.7.4.6 ß-Ureido (NH ) 4.2.99.2 H C

CH-COO 1.2.1.11 OH

CHO

1.1.1.31 4.6.1.1 NHCOCH CH NH H CC 1.14.17.1 4.3.2.1 2 CH CH NH N H 2 2 + CH COO 2 3 3.5.1.2 6.3.1.2 Carnosine + 23 NAD( NH + NH 2.7.3.2 2 aspartate O COO

OOC CO 22 CH CHCOO 4.4.1.15 NH 5-Hydroxy- Dehydro- - Carbamoyl NH shikimate tryptophan 3 OC OH OH HISTAMINE C NH C NH 32 C NH CITRULLINE OCH 23 2 CH 23 23

3 3-Amino-

2.3.1.30 CH CH COSCoA 2.1.3.1 P HSO ACID HO 1.4.1.14 4.1.1.41 5.1.99.1 4-OH-3-Methoxy- POOL

Cyclic AMP Cyclic quinate FOLIC 1.13.11.5

+ 4 2 (Noradrenaline) 2.1.1.6 O PO NH O 2.1.3.3 O Dehydro- Propanoyl-CoA Methylmalonyl-CoA 2.7.2.4 Cat. No. M3782 Cat. No. NH semialdehyde Methylmalonyl OOC-CH-COSCoA 3.5.3.4 CH COO P Norepinephrine semi- 2.6.1.18 O TRYPTOPHAN 2.3.1.16 R 2.1.1.2 4.1.1.11 O Malonic 4.2.1.22 OHCCH 2 2 NH ARGININE aldehyde OOCCH CH CH GLUTAMATE 22 COO CHO H NCOO 3 Indole Indoxyl 3 + ORNITHINE O NH COO NH

NH 4.6.1.3 2 222 22 Urea R-CO-COO + 2.1.3.2 2 222 6.3.4.16 Maleyl H NCH CH CH CH OCH Carbamoyl- (NH H NCONH 4.1.2.5 H NCNHCH CH CH CH COO 1.4.4.2

4.1.99.1 1.4.1.19 2.1.4.1 2 2-OXO ACID 2-OXO 2.1.1.28 Formyl 4.1.1.28 OC 3 OH HC

CHOHCH NH

acetoacetate 1.6.6.4 6.3.5.4

2 1.18.6.1 + O 1.7.7.1 1.4.1.2 2 I + 222 2 2

2 T 2.1.2.1 CH CHO - 5.2.1.2 CH COO glycinamide-R 2 - A Glycine 2 (Normetadrenaline) 2 N Asparagine CH CH NH 3.1.3.3 23 2.6.1.- R-CH(NH ) CH COCOO NH

22 1.2.1.16 N 2.1.2.2 OH 4.1.1.12 1.2.1.18 I 3 23 22 CH COO + CH NH COO NH + Normetepinephrine O H NOCCH CH

4.3.1.1 M 22 ASPARTATE SERINE 4.1.1.71 2-AMINO ACID + 1.2.1.4 (Auxin)

NH 2 5.1.99.1 5.4.99.2

NH A NO H NCNHCH COO C OH 1.4.3.4 + 33 Indole-

3 S 2.6.1.52 N NO 23 OC 33 1.5.99.2 2.1.1.5 A O Guanidoacetate HOCH CH(NH )COO 2

2 33 2 32 GLYCINE (Adrenaline)

2.4.1.22 aldehyde acetoacetate CHOLINE 1.4.1.10 1.1.1.29 pyruvate

β OH

0 O OPPU P COO COO - e Glycine

s F 4.2.1.3 OH OH 2 2 + O

a T

CO.SCoA H 2 α - O

OH h s

6.3.4.13 3 3

2.3.1.37 t 2 t +

A CH C(OH)COO CH i Glyoxylate n 1

H COO

1.4.1.1 2 CH OH y n OH - 2 OH CH OH

s F CH OH 4.4.1.15 - u 2 α

OH XO- 1.1.1.41 - CH OH

P ε

3.2.1.23 O CH 2.7.1.38 +

1.2.4.2 2.3.1.61 b OH c 4.1.1.1 4

2 T NH

H 4.1.3.7 HO HO O P COO u 3

4.1.1.32 A . 2

2 s 5-Amino- levulinate HO

1 CITRATE

O - +

COO . γ

OH g γ

OH c

ATP OH 2-O 6

CH δ +

CH(OH)COO CHCOO CH C

- . n ADP +

OOCCH i i P

6.3.4.7

O - + + 3 t 0 1.1.1.95 ISOCITRATE 6.2.1.4 ATP H 2.4.2.14

2 r OH GLUTARATE P 1 2H 2.7.1.6

H H OH HO 2H o 2 OH

GTP amine

2.7.7.10 2 1 COO p 2 + + oscp P

O 4.1.3.8 GDP

+ + OH s 2 H

OOCCOCH β + 2.7.1.2 2.7.1.1

CH O CO O ATP - OH n 2

OH P -Ribosyl SUCCINYL-CoA ADP

CH OH CH OH 2 2 2 α CH OH

H H

LACTOSE a

GALACTOSE

OPPU

HOH β 2 r CH

ADP P Galactose- P NAD+ t 5.1.3.2

CoA - 2 a OH

ATP β CH OH CH O 4.1.3.1

O + Glycerate CH(OH) -

Pi HO UDP -Galactose D + OH CH OH

H A

CO NADH P N HO + - 2 HOCH 2.7.1.11 _ OH + GLUCOSE HO

P P

2.7.7.12 6 O H CO +

H - OOCCHO - I OH Glyoxylate Cycle 1.8.1.4 H 1.2.4.1 CH OH 2.7.1.31 COO F HO 2.3.1.12 O COO COSCoA COO OOCCH

H + T - 3 P 3.1.3.9 δ O H 1.2.1.12 HO

H C HO + P H COCOO HO CH UQ . 2

1e-

3 2 A i - H SUCCINATE H CHOHCOO H

OH 2 E Pi P 2 4.1.3.2 5.4.2.2 2 H CCC OH OH 2.7.7.9 CH OH 2 CH 1.2.4.1 2.3.1.12 3.1.3.43 2.4.1.11 2.4.1.21 1e- ATP

UQ R 2 CO.SCoA COO 2 H CCC OH OH 2 5.3.1.9 2 HO 3.1.3.11 4.1.2.13 + OCH OCH OCH CHOH

ACETYL C 5.4.2.1 2.7.1.40 PYRUVATE CH =C(O ) Pi CH(O ) CHOHCHO UQ H 1:6-bis- P I UDP -Glucose P P P 2 Fructose- HOCH 4.2.1.2 4.2.1.11 Cyt.b OCH Glucose-6- P 1.3.5.1

N OOCCH=CHCOO + 2

FAD ADP + ADP ADP - P etc. PYRUVATE 2.7.2.3

or O NAD H 2- P -Glycerate 3- P -Glycerate 2.4.1.1 OCH P Glucose-1- P 1.1.1.37 + + + ADP + P

1.2.1.13 G P -enolpyruvate 2H ATP 2H CH(OH)CH Fructose-6- P 2 III OOCCOCH

3 R - OXALOACETATE 2H COO L FUMARATE 6.4.1.1 ) 4H 2H 2H 1:3-bis- P -Glycerate CH O

II E MALATE 3- P -Glyceraldehyde FAD CH P ADP 2.7.7.27 4.1.1.9 ATP D 3 1.1.1.39 Fe-S 2

+ N CO 2.4.1.21 ADP - .

_ + 1.1.1.49 2 5.3.1.8

Cyt.b CH(O

2 2.6.1.16 5.3.1.1 2 E 2e- + 2 IV Cyt.b 2.7.1.28 Glucose P

A H O I + 2.7.7.34 NH OH 2 2

2 2.6.1.44

2 XY

3.7.1.2 1.9.3.1 glycerate CO O 5.3.1.1 ATP OCH PP 1 NAD H 2UQ

Cu NADP 3.2.1.48 CHO 2UQ 2 OH 2.2.1.2

CH O 1.1.1.21 PP 2 UQ H 2.2.1.1 2.4.1.29 NAD UQ H

A Heme a OO H 2 H 2, 3-Diphospho- 2, 1.6. 5.3 OH H C OPPT O UQ H

B FMN H OPPT

HO OH 2Fe -S -S 2Fe 1.10.2.2 2e-

HOOCCH CO-SCoA 2.7.7.24 2.6.1.4 OH 3 O Malonyl-Co-A Heme a NADH+H Cyt.c Fe-S

Cu PROTONS 2e- OH OH OH O H CC 2 H C CC OH OH I 1.10.2.2 OH 2.3.1.38 / P O O 3 (5 Clusters)

CH CO-S-ACP

H+H NAD 1 Glucosamine-6- P 2.2.1.1 GDP-Glucose P FMN O 1.6.5.3 2.2.1.1 OH 2 3 D OH

2.3.1.39

UQ 4.1.3.4

2 1.1.1.22 4.2.1.18 3.2.1.26 2 CH OH 2 2 OH E lactone CH CH O COO Acetyl-ACP T 4.1.2.- + O P 2.4.1.13 OH OCH + + P -Glucono CH O

2 A Cyt.c OCH P P-Ribosyl- Sedoheptulose- PP O HO

Glyceraldehyde C P P Erythrose-4- LO HO TRANS 2H 4H 4H TDP -4-Oxo- HOOCCH CO-S-ACP NADPH COCH Malonyl-ACP

2.3.1.4 2 4.2.1.46 2

4.1.3.5 TDP -Glucose NADPH TDP -Rhamnose CH OH 6-deoxyglucose 2.3.1.16 5.3.1.8

5.1.3.13 2.3.1.16 PP OH O-ANTIGENS STARCH GLYCOGEN 2.7.6.1 2 2.7.1.4 P ) (Glycerone- Edition Designed by Donald E. Nicholson, D.Sc., HOCH PP OH 2.3.1.41 PP 2.3.1.41 2.3.1.41 CO 1.1.1.30 acetone- P 2.4.1.9 Dihydroxy- P CH 4.2.1.24 2.3.1.41 O O INULIN CELLULOSE PP

OH 3.1.1.17 OH C CHO H nd 2 2 2 OH 2.3.1.9 O + SUCROSE OHHO OH C=O H 32 CH CH O CH O - COO- 2 2 2 2 5-Amino- levulinate 5.5.1.4 2 - C 3 2 (C30) H CC OH H (C15) 2.7.1.7 2 4.1.3.5 2.7.4.2 2.5.1.1 2.5.1.21 2.7.1.36 2 5.1.3.1 1.1.1.32 2 (C10) 4.1.1.33 2 1.1.1.34 2.5.1.10 CH CH(OH)CH COO OH HO CH OH CH COO 2 CH CH The The University of England Leeds, – and Sigma-Aldrich 22 CH H CC OH OH 2 2 HO (C5) 3-OH-Butyrate 2 COCH COSACP 32 CH COO COO COCH COSACP 22 CH COO Squalene COO NCH CH COO 32 CH OH 3 22 mevalonate Diphospho- NADP COCH COSACP 2 3 22 2 32 CH OH Mevaldate Thromboxane B2 Thromboxane CH HO N H 3 2 14 2 HO CH C(OH)CH CHO Mevalonate 3 22 5.1.3.1 H H OH PP Farnesyl- PP Geranyl- CH C= CHCH O D-Ribose-5- P GDP - CH COCH COO glutaryl-CoA MANNOSE CH C(OH)CH CH OH D-Xylulose-5- P OCH 1.1.1.132 OH CH C(OH)CH CH O CH C(OH)CH COSCoA C H COCH COSACP Mitochondrial CH (CH ) COCH COS-CoA P Oxostearoyl-CoA ß-OH-ß-Methyl- CH C-CH H O 5.4.2.8 Acetoacetate 3 3 OCH 2 3 2n 2 OH P 2 3 22 2 2 Mannose-6- P 2 3 26 OH C 2 PP Isopentenyl- ALGINATES HC CH H CH OH HC KETONE BODIES KETONE 4.1.1.4 HN Mannuronate CH (CH ) OOC - + 33 CH (CH ) 3 H OH 3-Oxoacyl-ACP CH OH 5.4.99.7 COO CH (CH ) 2.4.1.33 1.14.99.7 OH n OH H H C COH OH CO Oxalate CH COCH HOOC-COOH OPPG 3 CH 5.3.99.5 Galactose 32 Acetone P P H CCCC OH H 32 O NHCOCH + 6- P -Gluconate 2 2 2 33 3-Oxo-Hexanoyl-ACP 1.2.3.5 O (C20) OH H 2.3.1.6 3-Oxo-Decanoyl-ACP 1.1.1.44 2 PP H CC OPPG NH 2 2 CH COCH COSCoA P 2 32 2 - Sorbitol 2.7.7.13 CH C= CHCH 2 - HO 3 H OH Mannose-1- P 3 22 2 Galactose 32 2 O O 2 3.1.2.11 2 2 2 OH COO 2 2 CH O CH O 3 PP CHOLINE 2 CH CH COCH COSCoA 3 HO OCH 3-Oxoacyl-CoA OH CH 2 O 22 CH (CH ) COCH COSCoA CH (CH ) COCH COSCoA 4.3.1.8 HOCH CH N(CH ) 2 H OH 2 4.2.1.75 COO CH P 1.1.1.100 CH OCH COO 2 CH Acetoacetyl-CoA - CH 5.3.1.6 3-Oxohexanoyl-CoA NH (Vitamin E) + P OH 2 3 2 2 33 1.1.1.8 CH OH 2 HO 2 2 OHCCOO CH Acyl-CoA HOCH HO Glycol HO CH O 3-Oxopentanoyl-CoA (C5) 1.1.1.100 HOCH CHO H OH C C C CO 3 P D-Ribulose-5- 3 O OH HO HOCH COO 3 Acetylcholine 1.1.1.100 CH OH CH COCH CH N(CH )

1.1.1.100 3.1.4.2 α -Tocopherol Phytol NHCOR aldehyde COO CH HOCH CH Glycolate N-Ac-Glucosamine-6- P CH CH COO HO CO

2.5.1.29 2.7.1.3 1.2.1.21 1.1.1.14 Glyoxylate 2.7.8.5 1.1.1.79 2 CH 1.4.3.8 OH P CH H CH 32 N N Fructose 2.4.1.69 2.4.1.68 3 2.7.1.32 HO H H Ethanolamine Fixation Ribulose-1,5-bis-P OH 2 2 2 2 Endoplasmic Reticulum CH C = CHCH O H OH

Inositol- P i HO 2.4.1.23 OH 3 H OH OH CCC w 5.1.3.4 32 2 α H D-Ribose 2.4.1.47 22 223 CH (CH ) CH=CHCH(OH)CHCH O-

Galacturonate o 1 P 2.7.1.17 HO ATP l CH OH 3.6.1.34 O α CH OH

i 2.5.1.32 R’-CO-OCH f + 1.13.11.34 ADP NH CH CH(OH)CH COS-ACP 2 2 3 2 14 3 22 H + 3-OH-Acyl-ACP 3 2n ATP OH CH CH N(CH ) 3 26 n 4.1.1.37 P - OPPU n 2.7.7.15 HOCH H + COO - 3 glycerol o + +

) 1.3.99.7 CH (CH ) CH(OH)CH COS-CoA CH (CH ) CH(OH)CH COS 2 2 - CH (CH ) O OH t O HOCH (C40) + CH (CH ) r synthase ATP 3 + - CH (CH )

O 5.3.99.3 O n n O g

t H 3-OH-Hexanoyl-ACP H 22 (Vitamin K) O H 3-OH-Decanoyl-ACP Glycerol O OH C CH CO CO CO NADP H+H H CDP-diacyl e H 2.7.1.47 H 2.7.1.107 NH +

HOCH c r CH CH O-CO-R COO H 2 2 2 2 + O-PO 2 r 2.3.1.15 2 2 2 CH 3.1.3.25

1.14.99.1 2

UDP -Glucuronate e

UDP-Galactose

Phytoene 3 u 3 2 3 l H 2 2 CH CHOHCH OH 2 2 OPPU

COO c OH 3.1.4.3 3.2.1.46 OCH CH 32 2 CH OH CH OH CH OH Phylloquinone CH CH O POCMP CH OPO CH O-CO-R 5.1.3.12 OH CCCCCO H CH OH e 2 CH

c CH CH CH(OH)CH COSCoA 3 22 3 P 3-OH-Acyl-CoA

ASCORBATE i UDP - CH + 8 O

γ 8 OH

r ε

ε CH CH COO 3.1.4.12 OH t

c HOCH CH CH CH(OH)CH COSCoA

i c Dehydroascorbate 2 O O

CH (CH ) CH(OH)CH COSCoA N CH (CH CH(OH)CH COSCoA l N

3.1.3.4

4.2.1.60 3

(Coenzyme Q) HO e 3-OH-Butanoyl-CoA Iduronate HO HOCH

Ubiquinone Menaquinone Plastoquinone H 4.2.1.60 4.2.1.61 l

+ 4.2.1.60

Ethanolamine- P c 2 2 N-Ac-Glucosamine-1- P H + -CO-OCH 3-OH-Pentanoyl-CoA

' CH OH +

H e 4.2.1.59 OH 2 2 -CO-OCH 4.2.1.58 NHCOR

2 2 33

( y Glycerophosphocholine + H COO R 3 CH Phosphatidylglycerol

C C H H H

R H c + CH O-CO-R CH OH

OH a

H 3.5.1.23 OH - H H + - CH CH N(CH ) OH O OH O HOCH H -- +

CO-S-ACP OH OH

HO H Serine Inositol H n 2 2 2.7.7.23

2 2 H

CHO N N o c Arachidonate Inositol 3 3

COO OH CCCO HH 4-Sphingenin - OH C CH CO OH OPPU VISION H C COH C CHO OH

CH COO COO CH H CCCO OH H 2 HO N H 2 4.2.1.17 CH CH COO 4.2.1.55 4.2.1.17 3 +

CDP-choline 2.7.7.14 2.4.1.17 2.7.8.3 R’-CO-OCH CH O CH O C PP -O O 2 H NHCOCH 2 2 hv 2 2 Diacyl CH OH H CH CH=CHCO.S-ACP D-Xylose COO H C COH OH CO HC glycerol D-Xylulose 3 2 12 2 2 + R-CH 3

PC + - + HO Rhodopsin CH CH=CHCO-S-ACP 3 NH 2 2

h 11- cis -Retinal + COO OH Palmitoyl-CoA 3 2 14 n 3 2 2 3 26 2.4.1.16 3 22 3 2n + Crotonoyl-ACP 3-OH-Butanoyl-ACP Acetoacetyl-ACP CH 23 p H + 3 25 + OH C CH OH CO +

PC H H www.iubmb.or 2 2 33 NH OPPU s PQ O + H 2, 3-Enoyl-ACP 2, CH O-CO-R CH O-CO-R" OH C CHH C CO CO OH OH 3.1.1.32 OPPU H CH (CH ) CH=CHCOS-CoA H 3 H CH CH (CH ) CH CH COO Palmitoleoyl-ACP CH (CH ) CH=CHCO-S-ACP o CH (CH ) CHITIN CHONDROITIN 2 O CH (CH ) 3, 4-Decenoyl-ACP 3, 22 1.1.1.9 + NHAC - O -

2, 3-Hexenoyl-ACP 2, h 32 CH CH N(CH ) NHAC + Lysolecithin N 2, 3-Decenoyl-ACP 2, N FAT

3.1.4.12 Cyt.f OH H CH CH=CHCOSCoA 2 2 3.1.2.20

4.1.3.20 3 22

32 p -Linolenate O 1e + H H H O H H 2 OH OH OH HYALURONIC ACIDHYALURONIC DERMATAN 2PQ

- COO

- o Dark - 2 2 Light CH CH CH=CHCOSCoA

- γ

H Crotonoyl-CoA t 3-Enoyl-CoA 2, 5.2.1.7 Pentenoyl-CoA 222 Opsin OCH CH O 5.2.1.3 O R’-CO-OCH CH OH O CH (CH ) CH=CHCOSCoA CH (CH ) CH=CHCOSCoA H C C H

2, 3-Dioxogulonate 2, O o 2 2 33 2 2e Xylitol 2e + HOCH 2e + 2 CH OH OPPU H CCC OH OH

- 2 h + 2, 3-Hexenoyl-CoA2, 3-OH-Hexanoyl-CoA OH CCCO H OH 1.3.1.10 _ OH C CHH CO H C COH C CHO H . H - 1.3.1.9 H HO H C H

COO HOCH Triacylglycerol

3.1.1.3 2 P 2 2 O CH CH N(CH ) CH O-CO-R CH CHNH 2 - CH O PO H HO SERINE + N N -

Cyt bc 1.3.1.9 2H

- CH OPO 2e 2 Cyt bf 2 Glucuronate HO CH O-CO-R CPP- 2 c CHO

2 3 1.3.1.9

COO- UDP -N-Ac- PQ 1e 2 i LECITHIN

2.7.8.3 L-Xylulose

+ 1.3.1.9 + O O O l O CH 2 Cardiolipin CH CH CH COO CH OH

_ L-RibuloseP L-Ribulose-5- Fe-S

UDP-Sugars . HOCH CH c

NHAC H H D-Arabinose D-Ribulose

- - OH O-CO-R CH CH y 3 2 14

2 CH OH 2 2

5.1.3.14 2.4.1.62 - UDP -N-Ac- OH 6.3.2.13 PQ PO + O O + CHOLESTEROL 1.1.1.102 1.1.1.19 GLYCAN Protoporphyrinogen Coproporphyrinogen Uroporphyrinogen Porphobilinogen 6.3.2.7-10 PEPTIDO- Glucosamine 2H THYLAKOID LUMEN H H -CO-OCH 1.3.99.7 ' 1.1.3.8 PQ H (C40) O UDP -N-Ac-Muramate HO 2 2PQ H R THYLAKOID MEMBRANE COO 2.7.8.1 (C40) OH OH (Vitamin A) 2 R'-CO-OCH Metarhodopsin Protons from Water 1.1.1.105 2 4.1.1.34 3 Translocated protons Translocated trans -Retinal 2 + + CHLOROPLAST OUTER MEMBRANE CHLOROPLAST OH 2.3.1.7 1.3.1.35 5.3.1.4 trans -Retinol 2 CH OH 3 CH CH COSACP H OH H CH - CH CH COSACP CH H NHAcyl PQ H CH - 3 OH H 1.3.1.35 3 2 2 n+ + OH OH NH CH 3.1.3.29 4.1.3.20 HO 2.7.1.60 2.7.7.43 H H H 3.1.3.29 OH 1.3.3.4 2.1.1.71 2 - 1.13.11.21 4.99.1.1 + NH HH OH CCCCCO H HH OH C C CO C 2.1.1.17 COO CH CH 2.4.99.7 CH 3 2 14 + O COO 322 (Sialate) OH 4.1.1.65 OH - OH OH H 2 3 26 2 2 OH 3 22 2 2 N O COO N Oleoyl-CoA (Cytosol) - Linoleate OH 22 H OH H n H OH H OH OH 2 A CH (CH ) COS-ACP B OPC 32 O Decanoyl-ACP Hexanoyl-ACP Butanoyl-ACP ACYL-ACP Palmitoyl-ACP CO P CH CH CH COSACP Stearoyl-CoA Dehydrostearoyl-CoA OH-Stearoyl-CoA O + Lycopene CH (CH ) Q Q 322 CH (CH ) 2e 2 2 33 Mn 2e 2 PHOTO- SYSTEM II O 2 ACNH H C C H CH (CH ) COS-CoA MUCINS P680 Gulonate OH CCCC H HH Chl.a H - Fe O 3222 OH CCC HOH OH C CH C CHO OH OH C CH C CHO OH ACNH OCH CH ACYL-CoA OH C CHH C CHO - O HOCH CH CH CH COSCoA O - OH 3 22 2 2 CH CH N(CH ) + CH OH OH HOCH 3222 Butanoyl-CoA CH O Hexanoyl-CoA 2 3.1.1.28 O-Acyl-carnitine 2 2 2 OH O O 2 2 Pheophytin 2 CH CH CH CH COSCoA O-Acyl-carnitine Pentanoyl-CoA 3 2 14 N-Ac-Neuraminate 2 2 - H N N 2 (Mitochondria) 2 2 ACYL-CoA CH (CH ) CH CH COSCoA Odd C Fatty acids C Fatty Odd O O O CH (CH ) CH CH COSCoA 2 L-Lyxose CH CH CH COO HO CH Gulonolactone 2-Oxogulonolactone 2 HEME CH OH 3-Dehydrogulonate inositol CHOH CHOH HO L-Xylose CH (CH ) COCHCH OH Serine ß-CAROTENE 2 L-Arabitol CH CHOH CHOH CH O-CO-R HEMOGLOBIN CH O-PO 2 2 HO Choline 1.14.99.5 CEPHALIN L-Arabinose HO Carnitine CH O-CO-R CH O P HOCH Retinoate neuraminate STEROIDS HOCH 1.2.1.36 3.1.1.21 HOCH 2.3.1.76 HC HOCH HOCH CMP -N-Acetyl Phosphatidyl H N-Ac-Mannosamine Phosphatidyl CH OPO -CO-OCH ethanolamine CH OCH=CHR SPHINGOMYELIN ' 3 3 Progesterone 2.3.1.50 3 2 12 Dehydrosphinganin Sphinganin Retinol esters plasmalogen Pregnenolone R Gangliosides GANGLIOSIDES HC HC 3.1.1.18 2003 International Union of Biochemistry and Molecular Biology AcNH 1.1.1.45 -CO-OCH AcNH CH (CH ) CH=CHCH(OH)CHCH O ' GLYCOPROTEINS

R N-Ac-Mannosamine-6- P R-CO-OCH © I I I I I I I I I I I I I I I T T T L T T L T L L P S Y E S S S P E S E X S E S P E S E S P S Y E S S E P S P P S S E S P P Y S P Y S E S P H N H R N D H N D B N H D R A D A N H H D R D R H R N A C C H A R D D O O O O O O O G O O O O O O 8

Advancements in Enzyme-Based Fuel Cell Batteries, Sensors and Emissions Reduction Strategies Dr. Shelley Minteer, Ph. D.

Advancements in Enzyme-Based Fuel Cell Batteries, Sensors and Emissions Reduction Strategies

Sigma® Life Science and Aldrich Materials In addition to synthetic polymer supports, Dr. Minteer’s lab has also utilized mitochondria Science merge to help researchers Dr. Minteer and her collaborator, Dr. Michael to generate current by metabolism of completely rethink tomorrow’s Cooney of the University of Hawaii, classic cellular energy sources. The resulting immobilized enzyme technologies. have immobilized dehydrogenases on electrons are shuffled through the electron macroporous chitosan scaffolds. The transport chain where they reduce oxygen Today’s fuel cells and batteries lack the scaffolds were fabricated from solutions at cytochrome c oxidase and combine with ability to produce electrical power for of native and hydrophobically modified the protons from oxidation to form water. the long periods of time we desire and chitosan polymers through the process During this metabolic process it is possible commonly incorporate toxic or precious of thermally induced phase separation. for cytochrome c to undergo direct electron metals to produce and store their energy. The hydrophobically modified chitosan transfer to a carbon electrode and complete Enzyme-based fuel cells have potentially is proposed to possess amphiphilic oxygen reduction at a platinum cathode. higher energy density than traditional micelles into which the enzyme can be Taking advantage of the fact that batteries using much more environmentally encapsulated and retained. nitroaromatics can decouple the inhibition compatible materials. While the idea One of Dr. Minteer’s basic biofuel cell of pyruvate metabolism, mitochondria- of enzyme-based biofuel cells is not a batteries employs an ethanol/alcohol modified electrodes have also been used new concept, recent developments have dehydrogenase system to generate a to electrochemically sense the presence of captured the interests of the research proton gradient across the conductive nitroaromatic explosive compounds. These community. Dr. Shelley Minteer at membrane. They have also refined these oligomycin inhibited mitochondria-modified Saint Louis University Department of dehydrogenase systems by replacing electrodes were employed as the bioanode in Chemistry is one of the pioneers in this area. traditional NAD(H) cofactor-dependent a pyruvate/air biofuel cell. This concept could Dr. Minteer and her team have developed dehydrogenases with pyrroloquinoline be used as a traditional electrochemical sensor a new enzyme immobilization technology quinone(PQQ)-dependent dehydrogenases or as a self-powered sensor. that has begun to revolutionize the way we to improve efficiency. Other multienzyme think about using enzymes for long-term Dr. Minteer's Nafion-based immobilization coupled systems use classical glycolytic catalytic applications. technology is also addressing the reduction of and Krebs cycle enzyme systems and fluegas emissions from coal-fired power plants Dr. Minteer’s enzyme immobilization corresponding metabolic substrates to using carbonic anhydrase to sequester CO . strategy encapsulates and stabilizes Sigma generate power. 2 enzymes in modified Nafion® polymer matrices from Aldrich. The efficacy of Dr. Minteer’s immobilized enzymes can be measured in years compared to days for Enzymes immobilized other biofuel cell technologies. Dr. Minteer in Nafion® membrane are encapsulated in has successfully demonstrated the use of the polymer resulting several metabolic fuels such as glycerol, in increased stability fatty acids, ethanol and other alcohols, while retaining active site availability and pyruvate and glucose to provide the fuel optimized orientation. source, while a wide variety of enzymes and coupled multienzyme systems provide the catalytic power to convert the fuel to electrical current. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 9

An an example of one of Dr Minteer’s fuel cells; Potentiostat utilizing pyruvate as the initial fuel source, this fuel cell incorporates a multi-enzyme coupled system that generates a proton gradient source of energy Pyruvate Deh. NADP+ analogous to that produced in vivo in the Kreb’s Cycle.

Acetyl CoA NADPH Platinum P Modified Nafion® Membr Citrate Synthase Carbon oly(meth

Citrate

vate Aconitase Par pe ylene green) Isocitrate Pyru Isocitrate Deh. O2 + CO NADP 2 α-Ketoglutarate NADPH α-Ketoglutarate Deh. Oxaloacetate NAD+ CO ane and Enz 2 Succinyl-CoA NADH Succinyl-CoA p Synthetase GDP GTP Succinate DehydrogenaseSuccinate ymes

FAD H O Fumarate FADH 2 2 Fuel Fumarase Solution Malate DehydrogenaseMalate NAD+ NADH

Cathode Anode NRE 212 Nafion®

References (1) S.D. Minteer, B.Y. Liaw, and M.J. Cooney, “Enzyme-based (10) A.E. Blackwell, M.J. Moehlenbrock, J.R. Worsham, and S.D. (19) M. J. Moehlenbrock, R. L. Arechederra, K. H. Sjoholm, biofuel cells,” Current Opinions in Biotechnology, 2007, 18, Minteer, “Comparison of Electropolymerized Thiazine and S.D. Minteer, “Analytical Techniques for Character- 228–234. Dyes as an Electrocatalyst in Enzymatic Biofuel Cells izing Enzymatic Biofuel Cells,” Analytical Chemistry, 2009, (2) C.M. Moore, N.L. Akers, and S.D. Minteer, “Improving the and Self Powered Sensors,” Journal of Nanoscience and 81(23), 9538–9545. Environment for Immobilized Dehydrogenase Enzymes Nanotechnology, 2009, 9, 1719–1726. (20) P. Addo, R. Arechederra, and S. D. Minteer, “Evaluating En- by Modifying Nafion with Tetraalkylammonium Bro- (11) M. Cooney, V. Svoboda, C. Lau, G. Martin, and S.D. Mint- zyme Cascades for Methanol/Air Biofuel Cells Based on mides,” Biomacromolecules, 5, 2004, 1241–1247. eer, “Enzymatic Biofuel Cells,” Energy and Environmental NAD-dependent Enzymes,” Electroanalysis, 2010, 22(7-8), (3) M.J. Cooney, M. Windmeisser, B.Y. Liaw, C. Lau, T. Klotz- Science, 2008, 1, 320–337. 807–812. bach, and S.D. Minteer, “Design of Chitosan Gel Pore (12) M.J. Cooney, J. Petermann, C. Lau, S.D. Minteer, “Charac- (21) R. Rincoln, M. Germain, K. Artyushkova, M. Mojica, M. Structure: Towards Enzyme Catalyzed Flow-Through terization and Evaluation of Hydrophobically Modified Germaine, P. Atanassov, and S.D. Minteer, “Structure and Electrodes,” Journal of Materials Chemistry, 2008, 18, Chitosan Scaffolds: towards design of enzyme immo- Electrochemical Properties of Electrocatalysts for NADH 667–674. bilized flow-through electrodes,” Carbohydrate Polymers, Oxidation,” Electroanalysis, 2010, 22(7–8), 799–806. (4) P. Atanassov, C. Apblett, S. Banta, S. Brozik, S. Calabrese 2009, 75, 428–435. (22) W. Gellett, M. Kesmez, J. Schumacher, N. Akers, and S.D. Barton, M. Cooney, B.Y. Liaw, S. Mukerjee, and S.D. (13) G. Martin, S. Minteer, and M.J. Cooney, “Spatial distribu- Minteer, “Biofuel Cells for Portable Power,” Electroanalysis, Minteer, “Enzymatic Biofuel Cells,” Electrochemical Society tion of malate dehydrogenase in amphiphilic chitosan 2010, 22(7–8), 727–731. Interface, 16, 2007, 28–31. scaffolds,” Applied Materials and Interfaces, 2009, 1(2) (23) D. Sokic-Lazic, R.L. Arechederra, B.L. Treu, and S.D. (5) T.L. Klotzbach, M. Watt, Y. Ansari, and S.D. Minteer, 367–372. Minteer, “Oxidation of Biofuels: Fuel Diversity and Ef- “Improving the microenvironment for enzyme immo- (14) C. Hettige, S.D. Minteer, and S. Calabrese Barton, “Simula- fectiveness of Fuel Oxidation through Multiple Enzyme bilization at electrodes by hydrophobically modifying tion of Multi-Step Enzyme Electrodes,” ECS Transactions, Cascades,” Electroanalysis, 2010, 22(7–8), 757–764. chitosan and Nafion polymers,” Journal of Membrane 2008, 13/21, 99–109. (24) C. Lau, G. Martin, S.D. Minteer, and M.J. Cooney, “Devel- Science, 2008, 311, 81–88. (15) K. Sjoholm, M.J. Cooney, and S.D. Minteer, “Effects of opment of a Chitosan Scaffold Electrode for Fuel Cell (6) V. Svoboda, M. Cooney, B.Y. Liaw, S. Minteer, E. Piles, D. Degree of Deacetylation on Enzyme Immobilization Applications,” Electroanalysis, 2010, 22(7–8), 793–798. Lehnert, S.C. Barton, R. Rincon, and P. Atanassov, “Stan- in Hydrophobically Modified Chitosan,” Carbohydrate (25) R. Arechederra, B.L. Treu, and S.D. Minteer, “Develop- dardized characterization of biocatalytic electrodes,” Polymers, 2009, 77, 420–424. ment of Glycerol/Oxygen Biofuel Cells,” Journal of Power Electroanalysis, 2008, 20, 1099–1109. (16) G. Martin, S.D. Minteer, and M.J. Cooney, “Fluorescence Sources, 173, 2007, 156–161. (7) B.L. Treu, R. Arechederra, and S.D. Minteer, “Bioelectroca- characterization of chemical microenvironments in hy- (26) R. Arechederra and S.D. Minteer, “Organelle-Based Bio- talysis of Ethanol via PQQ-Dependent Dehydrogenases drophobically modified chitosan,” Carbohydrate Polymers, fuel Cells: Immobilized Mitochondria at Carbon Paper Utilizing Carbon Nanomaterial Supports,” Journal of 2009, 77, 695–702. Electrodes,” Electrochimica Acta, 2008, 53, 6698–6703. Nanoscience and Nanotechnology, 2009, 9, 2374–2380. (17) D. Sokic-Lazic and S.D. Minteer, “Pyruvate/air enzymatic (27) M. Germain, R. Arechederra, and S.D. Minteer, “Nitroaro- (8) B.L. Treu and S.D. Minteer, “Isolation and Purification biofuel cell capable of complete oxidation,” Electrochemi- matic Actuation of Mitochondrial Bioelectrocatalysis for of PQQ-dependent Lactate Dehydrogenase from cal and Solid-State Letters, 2009, 12(9), F26–F28. Self Powered Explosive Sensors,” Journal of the American Gluconobacter and Use for Direct Electron Transfer at (18) M.C. Beilke, T.L. Klotzbach, B.L. Treu, D. Sokic-Lazic, J. Chemical Society, 2008, 130(46), 15272–15273. Carbon and Gold Electrodes,” Bioelectrochemistry, 2008, Wildrick, E.R. Amend, L.M. Gebhart, R.L. Arechederra, M.N. (28) R. Arechederra, K. Boehm, and S.D. Minteer, “ Mitochon- 74, 71–77. Germain, M.J. Moehlenbrock, Sudhanshu, and S.D. Mint- drial Bioelectrocatalysis for Biofuel Cell Applications,” (9) D. Sokic-Lazic and S.D. Minteer, “Citric acid cycle biomim- eer, “Enzymatic Biofuel Cells,” in Micro-Fuel Cells: Principles Electrochimica Acta, 2009, 54, 7268–7273. ic on a carbon electrode,” Biosensors and Bioelectronics, and Applications, Elsevier, 2009, 179–241. 2008, 24, 945–950. 10

Selected Enzymes for Biofuel Cell Research

Enzyme-based biofuel cells are incorporating Inhibitors: Compounds that react with free Alcohol Dehydrogenase, Deinococcus a wide variety of enzymes. Sigma-Aldrich sulfhydryls, including N-alkylmaleimides and radiodurans, recombinant from E. coli iodoacetamide. manufactures hundreds of enzymes, many ADH Zinc chelator inhibitors, including [9031‑72‑5] of which may have potential applications in 1,10-phenanthroline,  activity: ≥10000 units/mL biofuel cells. For a complete list of enzymes, 8-hydroxyquinoline, 2,2’-dipyridyl, and thiourea. One unit corresponds to the amount of data sheets, assay protocols, and other Substrate analogue inhibitors, including β-NAD enzyme which reduces 1 μmole ethyl-2-oxo-4- enzyme-related technical resources visit the analogs, purine and pyrimidine derivatives, phenylbutyrate per minute at pH 7.0 and 37 °C chloroethanol, and fluoroethanol. web-based Sigma Enzyme Explorer at sigma- 16892-1ML 1 mL 1% aldrich.com/enzymeexplorer. The Enzyme Extinction Coefficient: E = 14.6 (water, 280 nm) 16892-5ML 5 mL Explorer also features the IUBMB on-line One unit will convert 1.0 μmole of ethanol to acetaldehyde per min at pH 8.8 at 25 °C. Alcohol Dehydrogenase, NADP+ metabolic pathway chart with over 500 links to dependent from Thermoanaerobium enzymes, substrates and cofactors.  lyophilized powder (contains buffer salts), activity: brockii ≥300 units/mg protein Alcohol:NADP+ oxidoreductase; TBADH The following products are only a small sample Solids containing ≤ 2% citrate buffer salts suitable [9028‑12‑0] of the enzymes available from Sigma-Aldrich for conventional determination of β-NAD, β-NADH, ethanol and acetaldehyde An extremely thermostable enzyme with broad of potential use in biofuel cell research. substrate specificity for alcohols, ketones and Contains bound β-NAD and β-NADH and is not acetaldehyde. ▼ Alcohol Dehydrogenase suitable for the recycling microassay of β-NAD and β-NADH. If you require ADH for this purpose, see One unit will oxidize 1.0 μmole of 2-propanol to ADH Cat. No. A3263. acetone per min at pH 7.8 at 40 °C in the presence Alcohol Dehydrogenase from of NADP+. Parvibaculum lavamentivorans A7011-7.5KU 7500 units  lyophilized powder, activity: 5–15 units/mg A7011-15KU 15000 units  recombinant, expressed in Escherichia coli, activity: protein A7011-30KU 30000 units ≥40.0 units/mg Contains phosphate buffer salts and A7011-75KU 75000 units One unit corresponds to the amount of enzyme dithioerythritol A7011-150KU 150000 units which reduces 1 μmole ethyl acetate per minute A8435-100UN 100 units A7011-300KU 300000 units at pH 7.0 and 30 °C. A8435-250UN 250 units  activity: ≥300 units/mg protein 75449-1ML 1 mL  lyophilized powder, activity: 30–90 units/mg Solids containing <2% citrate buffer salts suitable 75449-5ML 5 mL protein for recycling micro-assay of β-NAD and β-NADH Purified Alcohol Dehydrogenase from A3263-7.5KU 7500 units Contains sodium citrate and dithioerythritol. Saccharomyces cerevisiae A3263-15KU 15000 units Alcohol:NAD+ oxidoreductase; Alcohol Dehydro A3263-30KU 30000 units A9287-100UN 100 units genase from yeast A3263-75KU 75000 units Alcohol Dehydrogenase ▲ [9031‑72‑5] A3263-150KU 150000 units A 141 kDa tetramer containing 4 equal subunits. Bilirubin Oxidase from Myrothecium The active site of each subunit contains a zinc verrucaria atom. Each active site also contains 2 reactive Bilirubin:oxygen oxidoreductase sulfhydryl groups and a histidine residue. [80619‑01‑8] Isoelectric point: 5.4–5.8  lyophilized powder, activity: 15–65 units/mg Optimal pH: 8.6–9.0 protein Substrates: Yeast ADH is most active with ethanol One unit will oxidize 1.0 μmole of bilirubin per min and its activity decreases as the size of the alcohol at pH 8.4 at 37 °C. increases or decreases. Branched chain alcohols B0390-25UN 25 units and secondary alcohols also have very low activity. B0390-100UN 100 units -3 KM (ethanol) = 2.1 × 10 M -1 KM (methanol = 1.3 × 10 M -1 KM (isopropanol) = 1.4 × 10 M Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 11

Cytochrome c Oxidase from bovine heart A thermostable enzyme with resistance to organic  lyophilized powder, activity: 100,000– Ferrocytochrome-c:oxygen oxidoreductase, Complex solvents. Enzyme also shows substantial activity 250,000 units/g solid (without added oxygen) IV; EC 1.9.3.1 with d-galactose as substrate. May contain traces of amylase, maltase, [9001‑16‑5] Suspension in 2.8 M (NH4)2SO4, 50 mM phosphate glycogenase, invertase, and galactose oxidase. Cytochrome c oxidase is the principal terminal buffer, pH 7.0 Protein determined by biuret oxidase of high oxygen affinity in the aerobic One unit will oxidize 1.0 μmole of d-glucose to metabolism of all animals, plants, yeasts and some d-glucono-δ-lactone per min at pH 7.0 at 37 °C in G7141-10KU 10000 units bacteria. + the presence of NADP . G7141-50KU 50000 units Supplied as a solution in 25 mM Tris-HCl buffer, G5909-100UN 100 units G7141-250KU 250000 units pH 7.8, 5 mM EDTA, and 39 mM sodium dodecyl G5909-500UN 500 units G7141-1MU 1000000 units maltoside. G7141-2.5MU 2500000 units activity: ≥20 units/mg protein Glucose Oxidase from Aspergillus niger One unit of cytochrome c oxidase will oxidize 1.0 ▼ Laccase G.Od.; GOx; β-d-Glucose:oxygen 1-oxidoreductase μmole of ferrocytochrome c per min. at 25 °C at [80498‑15‑3] [9001‑37‑0] pH 6.0. Laccase from Rhus vernicifera Molecular Weight: 160 kDa (gel filtration) C5499-250UG 250 μg Benzene diol:oxygen oxidoreductase pI: 4.2  crude acetone powder, activity: ≥50 units/mg 1% d-Fructose Dehydrogenase from Extinction coefficient: E = 16.7 (280 nm) solid Gluconobacter industrius Glucose oxidase from Aspergillus niger is a dimer One unit will produce a ΔA530 of 0.001 per min at d-Fructose:(acceptor) 5-oxidoreductase consisting of 2 equal subunits with a molecular pH 6.5 at 30 °C in a 3 mL reaction volume using [37250‑85‑4] mass of 80 kDa each. Each subunit contains one syringaldazine as substrate.  lyophilized powder, activity: 400–1,200 units/mg flavin adenine dinulceotide moiety and one iron. L2157-10KU 10000 units protein The enzyme is a glycoprotein containing ~16% Lyophilized powder containing citrate-phosphate neutral sugar and 2% amino sugars. The enzyme Laccase from Trametes versicolor buffer salts, TRITON® X-100, and stabilizer also contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. former nomenclature: Coriolus versicolor One unit will convert 1.0 μmole d-fructose to  powder, activity: >20 units/mg 5-ketofructose per min at pH 4.5 at 37 °C. Glucose oxidase is capable of oxidizing d-aldohexoses, monodeoxy-d-glucoses, and One unit corresponds to the amount of enzyme F5152-250UN 250 units methyl-d-glucoses at varying rates. which converts 1 μmole of catechol per minute at pH 4.5 and 25 °C Glucose Dehydrogenase from calf liver The pH optimum for glucose oxidase is 5.5, while it has a broad activity range of pH 4–7. Glucose 53739-100MG-F 100 mg β-d-Glucose: NAD[P]+ 1-oxidoreductase oxidase is specific for β-d-glucose with a K of 53739-1G-F 1 g [9028‑53‑9] M 33–110 mM.  powder, activity: ≥0.5 units/mg  activity: ~15 units/g solid Glucose oxidase does not require any Crude powder One unit corresponds to the amount of enzyme activators, but it is inhibited by Ag+, which converts 1 μmole of catechol per minute at One unit will oxidize 1.0 μmole of β-d-glucose to Hg2+, Cu2+, phenylmercuric acetate, and pH 6.0 and 25 °C d-glucono-δ-lactone per min at pH 7.6 at 25 °C. p-chloromercuribenzoate. It is not inhibited by 38429-1G 1 g G5625 Inquire the nonmetallic SH reagents: N-ethylmaleimide, 38429-10G 10 g iodoacetate, and iodoacetamide. ▲ Glucose Dehydrogenase from Glucose oxidase can be utilized in the enzymatic Laccase Pseudomonas sp. determination of d-glucose in solution. As glucose [9028‑53‑9] oxidase oxidizes β-d-glucose to d-gluconolactate  powder, activity: ≥200 units/mg and hydrogen peroxide, horseradish peroxidase One unit corresponds to the amount of enzyme is often used as the coupling enzyme for glucose which will oxidizes 1 μmole β-D-glucose to determination. Although glucose oxidase is D-glucono-δ-lactone per minute at pH 8.0 and specific for β-d-glucose, solutions of d-glucose can 37 °C be quantified as α-d-glucose will mutorotate to β-d-glucose as the β-d-glucose is consumed by 19359-10MG-F 10 mg the enzymatic reaction. Glucose Dehydrogenase from One unit will oxidize 1.0 μmole of β-d-glucose to d Thermoplasma acidophilum -gluconolactone and H2O2 per min at pH 5.1 at 35 °C, equivalent to an O uptake of 22.4 μl per min. If β-D-Glucose:NAD[P]+ 1-oxidoreductase 2 [9028‑53‑9] the reaction mixture is saturated with oxygen, the activity may increase by up to 100%.  recombinant, expressed in Escherichia coli, ammonium sulfate suspension, activity: ≥100 units/mg protein (glucose substrate), activity: ≥50 units/mg protein (galactose substrate) 12

Enzymes and Reagents for Ethanol Research Enzymes for Lignocellulosic Ethanol Research

Enzymes and Reagents for Ethanol Research

Enzymes for Lignocellulosic How do we physically dismantle biomass penetrate the barrier posed by the closely Ethanol Research on a molecular level to yield cellulosic integrated network of lignins surrounding polymers available to hydrolysis by cellulase cellulose microfibrils? Sigma-Aldrich is committed to helping and other glycolytic enzymes? How do we unlock today's challenge of releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. To the Plant Cell Wall untrained eye, starch and cellulose structures appear almost identical. Yet the barriers Middle between cellulosic biomass hydrolysis and Lamella ethanol production appear have proven to Pectin be orders of magnitude more challenging Cell Wall than with starch derived biomass. Hemicellulose Although many of the biochemical aspects Cellulose Microﬁbril of these challenges are well understood, Plasma they remain an enigma of nature. The two Membrane main, and closely related hindrances to the enzymatic hydrolysis of cellulosic polymers are:

Visit the Enzyme Explorer Assay Library

Features over 600 detailed procedures for measuring enzyme activity and related metabolites. The Library is the result of over ten years of in-house work on analytical procedures developed by Sigma-Aldrich® scientists.

Visit sigma.com/enzymeexplorer Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 13

Cellulases and Related Enzyme Activities

Cellulase catalyzes the endohydrolysis of β−(1→4)-d- glucosidic linkages in cellulose, lichenin and cereal β-d-glucans endo-1,4-β-D-Glucanase CH2OH O OH Most cellulase preparations contain various classes of OH CH2OH cellulases and related activities. O O OH The endo-β-(1→4)-d-glucanases (EGs) cleave Cellobiosidase II OH • CH2OH O cellulose in its internal non-terminal regions, yielding O OH oligosaccharides. OH CH2OH O O The two forms of cellobiohydrolases (CBH) are b-Glucosidase OH • OH considered exo-β-(1→4)-d-glucanases, releasing Cellobiosidase I CH2OH O cellobiose disaccharides from the cellulose chain OH O reducing end (CBHI), and non-reducing end (CBHII). OH CH2OH O O • β-Glucosidase is an exohydrolase that yields glucose OH OH monosaccharides from soluble oligosaccharaides such OH as cellobiose. OH Other activities that can be found in cellulase preparations include xylanase, hemicellulase, and laminarinase.

Cereal β-Glucan is a polymer of β-(1→4)- CH2OH d-glycopyranosyl units occupying as predominantly Laminaranase Cellulase O O cellotriose and cellotetraose separated by single Cellulase OH β-(1→3)-d-glycopyranosyl units. Crosslinking can occur CH OH CH OH 2 2 within the consecutive cellotriose regions. O O O O OH Cellulase OH CH2OH O O OH OH OH OH CH2OH b-(1-4)-D-glucose b-(1-3)-D-glucose O O OH O OH b-(1-4)-D-glucose

OH b-(1-4)-D-glucose

Hemicelluloses are a group of plant-derived heteropolysaccharides associated with cellulose and lignin. The most common hemicelluloses are xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan. In angiosperms, the principal hemicellulose component, xylan, is a polymer of β−(1→4)-d-xylopyranose. In arabinoxylan, O branching occurs at the C2 & C3 postions with a-L-arab- Xylanase OH O inofunaose. Glucuronoxylan, also found in angiosperms,

O O has the xylan backbone with 4-0 methylglucuronic acid OH branching. In addition, arabinose branching as well as acetylation may be present. Gymnosperms contain O O glucomannans comprised primarily of d-mannosyl and OH OH OH d-glucosyl residues.. O O O OH Hemicellulase preparations are typically a mixture of OH • glycolytic enzymes containing xylanase, mananase and

OH other activities. • Xylanase catalyzes the endohydrolysis of β−(1→4)- d-xylosidic linkages in xylans yielding various β−(1→4)- d-xylooligosaccharides. 14

Cellobiase from Aspergillus niger Cellulase from Trichoderma viride One unit will liberate 1.0 μmole of glucose Cellobiase enzyme preparation obtained by 1,4-(1,3:1,4)-β-D-Glucan 4-glucano-hydrolase from cellulose in one hr at pH 5.0 at 37 °C (2 hr submerged fermentation of an Aspergillus One unit will liberate 1.0 μmole of glucose from incubation time). niger microorganism. The cellobiase hydrolyzes cellulose in one hour at pH 5.0 at 37 °C (2 hr G4423-100G 100 g incubation time). cellobiose to glucose. β-Glucanase ▲  Novozyme 188 liquid, activity: ≥250 units/g  crude powder, activity: 3–10 units/mg solid β-(1→3)-d-Glucanase from Helix pomatia C9422-5KU 5000 units A product of Novozyme Corp. C9422-10KU 10000 units [9044‑93‑3] C6105-50ML 50 mL  powder, activity: 0.5–1.5 units/mg C6105-250ML 250 mL  Onozuka RS powder, activity: ≥5,000 units/g solid One unit corresponds to the amount of enzyme ▼ Cellulase Manufactured by Yakult which liberates 1 μmole of glucose from laminarin (Cat. No. 61340) per minute at pH 5.0 and 37 °C [9012‑54‑8] C0615-1G 1 g Improved filterability of wines by enzymic Cellulase from Aspergillus sp. Cellulase ▲ decomposition of carbohydrate-containing colloids1; Induction of hydrolases as a defense Carezyme 1000L® Driselase® from Basidiomycetes sp. reaction against pathogens, review2 activity: ≥1000 units/g [85186‑71‑6] Lit cited: 1. Wucherpfennig, K., and Dietrich, H. , Wein- produced by submerged fermentation of a Crude powder containing laminarinase, xylanase wirtschaft 118, 598 (1982); 2. Boller, T. , UCLA Symp. Mol. genetically modified Aspergillus microorganism and cellulase. Cell Biol., New Ser. 22, 247 (1985) A product of Novozyme Corp.  powder 49103-10MG 10 mg C2605-50ML 50 mL 49103-50MG 50 mg D9515-1G 1 g C2605-250ML 250 mL D9515-5G 5 g β-Glucosidase from almonds D9515-25G 25 g Cellulase from Trichoderma β-d-Glucoside glucohydrolase longibrachiatum [9001‑22‑3] ▼ β-Glucanase  lyophilized powder, activity: ≥2 units/mg solid  powder, activity: ≥1.0 unit/mg solid An acid cellulase with xylanase, pectinase, β-Glucanase from Aspergillus niger Crude mannanase, xyloglucanase, laminarase, [9074‑98‑0] One unit will liberate 1.0 μmole of glucose from β-glucosidase, β-xylosidase, α-L-  powder, activity: ~1 units/mg salicin per min at pH 5.0 at 37 °C. arabinofuranosidase, amylase, and protease One unit corresponds to the amount of enzyme G0395-2.5KU 2500 units activities which will release 1 μmole of reducing sugar G0395-5KU 5000 units One unit will liberate 1.0 μmole of glucose from equivalents (expressed as glucose) per minute at G0395-50KU 50000 units cellulose in one hour at pH 5.0 at 37 °C (2 hr pH 5.0 and 55 °C, using β-d-glucan  lyophilized powder, activity: 20–40 units/mg solid incubation time). (Cat. No. 49102) as substrate Chromatographically purified 49101-100MG 100 mg C9748-100G 100 g One unit will liberate 1.0 μmole of glucose from 49101-500MG 500 mg Cellulase from Trichoderma reesei salicin per min at pH 5.0 at 37 °C. ATCC 26921 β-Glucanase from Bacillus subtilis G4511-100UN 100 units G4511-250UN 250 units 1,4-(1,3:1,4)-β-D-Glucan 4-glucano-hydrolase [9074‑98‑0] G4511-1KU 1000 units  lyophilized powder, activity: ≥1 unit/mg solid  powder, activity: ~1 units/mg One unit will liberate 1.0 μmole of glucose One unit corresponds to the amount of Hemicellulase from Aspergillus niger from cellulose in one hr at pH 5.0 at 37 °C (2 hr enzyme which will release 1 μmol of reducing [9025‑56‑3] sugar equivalents (expressed as glucose) per incubation time).  powder, activity: 0.3–3.0 unit/mg solid (using a minute of pH 6.0 and 55 °C, using β-d-glucan β-galactose dehydrogenase system and locust C8546-2.5KU 2500 units (Cat. No. 49102) as substrate C8546-5KU 5000 units bean gum as substrate) 49106-100MG 100 mg C8546-10KU 10000 units An undefined mixture of glycolytic enzymes usually containing xylanase, mannanase and other  Celluclast® 1.5L aqueous solution, activity: β-Glucanase from Trichoderma activities. ≥700 units/g longibrachiatum One unit will produce a relative fluidity change Produced by submerged fermentation of a → A mixture composed mainly of β-1 3 / of 1 per 5 minutes using locust bean gum as selected strain of the fungus Trichoderma reesei → 1 4-glucanase, xylanase, and cellulase substrate at pH 4.5 at 40 °C and catalyzes the breakdown of cellulose into activities. β-glucosidase, β-xylosidase, α-L- glucose, cellobiose, and higher glucose polymers. arabinofuranosidase, H2125-150KU 150000 units A product of Novozyme Corp. amylase, and protease activities are also present. activity: ≥1.0 units/mg solid C2730-50ML 50 mL Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 15

▼ Laminarinase ▼ Xylanase Xylanase from Trichoderma viride endo-1,4-β-Xylanase; 1,4-β-d-Xylanxylanohydrolase Endo-1,3(4)-β-glucanase View more information on enzymes for complex [9025‑57‑4] [62213‑14‑3] carbohydrate analysis at sigma-aldrich.com/ enzymeexplorer  lyophilized powder, activity: 100–300 units/mg Laminarinase from Penicillium sp. protein 1,3-(1,3:1,4)-β-d-Glucan 3(4)-glucanohydrolase Xylanase from Thermomyces lanuginosus Contains sorbitol and sodium acetate buffer salts  lyophilized powder, activity: 5–10 units/mg [37278‑89‑0] One unit will liberate 1 μmole of reducing sugar protein Purified endo-β−(1→4)-xylanase from measured as xylose equivalents from xylan Lyophilized powder containing acetate buffer salts Thermomyces lanuginosus produced by submerged (Cat. No. X0627) per min at pH 4.5 at 30 °C. One unit will liberate 1.0 mg of reducing sugar fermentation of a genetically modified Aspergillus X3876-250UN 250 units (measured as glucose) from laminarin per min at oryzae microorganism X3876-1KU 1000 units pH 5.0 at 37 °C.  Pentopan Mono BG® powder, activity: Xylanase ▲ L9259-25UN 25 units ≥2500 units/g, recombinant, expressed in Aspergillus oryzae Laminarinase from Trichoderma sp. endo-1,4-β-Xylanase from Trichoderma A product of Novozyme Corp. longibrachiatum 1,3-[1,3;1,4]-β-d-Glucan 3(4)-glucanohydrolase X2753-10G 10 g 1,4-β-d-Xylanxylanohydrolase View more information on enzymes for complex X2753-50G 50 g Primary activity is an acid-neutral endo-1,4-β-d- carbohydrate analysis at sigma-aldrich.com/ xylanase, additional activities include β-glucanase, enzymeexplorer cellulase, pectinase, mannanase, xyloglucanase,  powder, activity: 100–400 units/g solid laminarase, β-glucosidase, β-xylosidase, α-L- Contains chitinase activity. arabinofuranosidase, amylase, and protease. One unit will liberate 1.0 mg of reducing sugar activity: ≥1.0 units/mg solid (measured as glucose) from laminarin per min at One unit will liberate 1 μmole of reducing sugar pH 5.0 at 37 °C. measured as xylose equivalents from xylan L5272-5UN 5 units (Cat. No. X0627) per min at pH 4.5 at 30 °C. L5272-25UN 25 units X2629-100G 100 g Laminarinase ▲

Online Metabolomics Resource Use the IUBMB–Sigma-Aldrich Interactive Metabolic Pathway Chart to find the Metabolite Standards you need. http://www.sigma-aldrich.com/ProductLookup.html? ProdNo=C7352&Brand=SIGMA The Metabolic Pathways Map contains over 500 hyperlinks to Sigma product listings. Just click on the metabolite name or the enzyme’s E.C. number to access product information.

You can access the chart at sigma-aldrich.com/metpath 16

Enzymes for Pectin Hydroylsis Pectins are complex-branched heteropolysaccharides primarily containing an α-(1→4) polygalacturonic acid backbone which can be randomly acetylated and methylated. Three different pectins have been isolated from plant cell walls.

Homogalacturonan is a composed of a poly-α−(1→4)- galacturonic acid backbone with random partial Pectinase Pectinase methylation and acetylation.

COCH 3 COOH COOH COCH 3 O O O O

OH O OH O OH O OH O

OH OH OOCH3 OH

Endo-pectin Lyase Pectinesterase

Oligo-a-(1-3)-D-arabinose branching Rhamnogalacturonan I is composed of alternating

CH2OH O OH O α−(1→2)-l-rhamnosyl-α−(1→4)-d-galacturonosyl CH2OH O OH CH 3 O CH2OH O backbone with two types of branching composed of a OH COCH 3 O O OH ribofuranose or galactose oligomers. OH O O O Oligo-b-(1-4)-D-galactose branching CH 3 O OH O HO

CH2OH CH2OH CH2OH COCH 3 OH OH HO O OOO O O O O O OH OH OH CH 3 O OH

OH OH OH COOH OH OH O OH O O OH α-(1-4)-D-galacturonic acid CH 3 O

b OH -Galactosidase OH O a-(1-2)-L-rhamnose

Rhamnogalacturonan II is composed of poly-α−(1→4)- d-galacturonic acid backbone with random partial methylation, acetylation and four types of branching. β-Arabinose α-Rhamnose

5 5 β-3-deoxy-D-lyxo- α-3-deoxy-D-manno- Pectinase 2-heptulosonic acid 2-octulosonic acid (Dha) (Kdo)

O O

COOCH 3 COOH COOH COOCH 3 COOCH 3 COOH COOH COOCH 3 O O O O O O O O O O OH O OOCCH3 O O OH OH O OH O OOCCH3 O

OH OH OH OH OOCCH3 OH O O

α-D-Xylose Pectinesterase β-D-Apiose 2 α-L-Rhamnose 2 α-L-Arabinopyra nose β-D-Apiose (2-O-methyl) 3' 3' β -L-Arabinofuranose 4 3 β-D-Galactose 2−α-L-Aceric(O-acetyl) 3−β-L-Rhamnose α-D-Galactose 2−β-L-Rhamnose 4 α-L-fucose 2 3 4

α -D-fucose β-D-Galactose β-D-Glucuronic Acid (2-O-methyl, 3-O-acetyl-) 2

α-D-Galactose Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 17

▼ Pectinase Pectolyase from Aspergillus japonicus  lyophilized powder, activity: ≥0.3 units/mg solid One unit will liberate 1.0 μmole of galacturonic acid Pectinase from Aspergillus aculeatus Reported to contain two types of pectinase, endopolygalacturonase (EC3.2.1.15), endopectin from polygalacturonic acid per min at pH 5.5 at  Pectinex® Ultra SPL aqueous solution, activity: lyase (EC4.2.2.10) and a maceration stimulating 25 °C. ≥9,500 units/mL factor. P3026-100MG 100 mg Highly active pectolytic enzyme preparation Used in plant protoplast preparation to digest cell P3026-250MG 250 mg produced by a selected strain of Aspergillus aculeatus wall prior to organelle isolation. P3026-1G 1 g A product of Novozyme Corp. P2611-50ML 50 mL P2611-250ML 250 mL Pectinase from Aspergillus niger Feeling the Heat? Then Capture It! Polygalacturonase solution from Aspergillus niger; Poly- (1,4-α-d-galacturonide) glycanohydrol ase [9032‑75‑1] Used in plant protoplast preparation to digest cell wall prior to organelle isolation.  aqueous glycerol solution, activity: ≥5 units/mg protein (Lowry) Solution in 40% glycerol One unit will liberate 1.0 μmole of galacturonic acid from polygalacturonic acid per min at pH 4.0 at 25 °C. P4716-5KU 5000 units P4716-10KU 10000 units P4716-25KU 25000 units P4716-100KU 100000 units

Pectinase from Aspergillus niger  Pectinex 3XL® aqueous solution Pectolytic enzyme preparation produced from a selected strain of Aspergillus niger: contains mainly pectintranseliminase, polygalacturonase, and pectinesterase and small amounts of hemicellulases and cellulases. A product of Novozyme Corp. P2736-50ML 50 mL P2736-250ML 250 mL

Pectinase from Rhizopus sp. Poly-(1,4-α-d-galacturonide) glycanohydrol ase; Poly Aldrich Materials Science — galacturonase We focus on materials so you can focus on results. [9032‑75‑1] Used in plant protoplast preparation to digest cell Materials for Renewable and Alternative wall prior to organelle isolation. Energy Solutions  Macerozyme R-10 powder, activity: • Solar Energy Conversion (Organic, Crystalline 400–800 units/g solid and Thin Film Photovoltaics) A source of pectinase activity, also containing cellulase and hemicellulase activities. • Hydrogen Economy One unit will liberate 1.0 μmole of galacturonic acid • Supercapacitors/Advanced Batteries from polygalacturonic acid per min at pH 4.0 at Alternative Fuels 25 °C. • Check out the Sigma-Aldrich Renewable & Alternative P2401-500UN 500 units Energy Web Portal: sigma-aldrich.com/renewable P2401-1KU 1000 units P2401-5KU 5000 units Pectinase ▲ sigma-aldrich.com 18

Enzymes for Lignin Depolymerization To some, lignin appears to be a foreign substance resembling a randomly modified polystyrene polymer, not resembling any of the more familiar biopolymers composed of carbohydrates, amino acids or nucleotides. However, next to cellulose, it is the second most abundant biopolymer on earth comprising as much as 30% Lignin Monomers of the dry mass of wood. Unlike other HO HO HO biopolymers, enzymatic degradation is oxidative versus hydrolytic and is limited to a select group of fungi and bacteria. Lignins appear to be heterogeneous and random CH3O CH3O OCH3 OH OH OH with regards to degree of polymerization, p-Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol branching, and monomer composition and sequence.

O CH3 Lignin O HO

OCH CH3O O 3 OH

O HO HO OH OH CH3O O H O HO OCH3 HO OH CH3O HO O O OH HO O O OCH3

CH O OCH OH OCH 3 O OCH O 3 O CH 3 O 3 3 O

OH CH3 O HO CH3O

O O CH3 OH O O O OH O

OH HO

CH3 O O OH

OH HO OH

O O

OCH3 OH

O HO

HO Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 19

▼ Laccase  powder, activity: ≥0.5 units/mg Manganese Peroxidase from One unit corresponds to the amount of enzyme Nematoloma frowardii [80498‑15‑3] which converts 1 μmole of catechol per minute at [114995‑15‑2] Laccase from Rhus vernicifera pH 6.0 and 25 °C 41563 Inquire Benzenediol:oxygen oxidoreductase 38429-1G 1 g  crude acetone powder, activity: ≥50 units/mg 38429-10G 10 g Manganese peroxidase from white-rot solid fungus (Phanerochaete chrysosporium) Laccase, Coriolus versicolor, CLEA One unit will produce a ΔA530 of 0.001 per min at MnP; Peroxidase, manganese; Manganese-dependent pH 6.5 at 30 °C in a 3 mL reaction volume using  Laccase, Coriolus versicolor, cross-linked enzyme lignin peroxidase syringaldazine as substrate. aggregate activity: ≥0.3 units/mg [114995‑15‑2] L2157-10KU 10000 units One unit corresponds to the amount of enzyme  powder, activity: ≥20 U/g which oxidizes 1 μmol ABTS per minute at pH 4.5 only partially soluble in water or buffer Laccase from Trametes versicolor and 25°C One unit corresponds to the amount of enzyme, former nomenclature: Coriolus versicolor 38837-100MG 100 mg which oxidizes 1 μmole Mn2+ per minute to Mn3+  powder, activity: >20 units/mg at pH 4.5 and 25 °C Laccase ▲ One unit corresponds to the amount of enzyme This product is supplied as a mixture of isozymes, which converts 1 μmole of catechol per minute at Lignin Peroxidase mol. mass (40–65 kDa). pH 4.5 and 25 °C LiP; Peroxidase, lignin; Ligninase 93014-10MG-F 10 mg 53739-100MG-F 100 mg [42613‑30‑9] 53739-1G-F 1 g  powder, activity: >0.1 units/mg One unit corresponds to the amount of enzyme, which oxidizes 1 μmole 3.4-dimethoxybenzyl alcohol per minute at pH 3.0 and 30 °C 42603-10MG-F 10 mg

Lignin peroxidase catalyzes the oxidative cleavage of carbon-carbon and ether bonds in lignin-related compounds. O O CH3O

CH3O

H O H O 2 2 2 O H HO OH OH HO

Lignin Peroxidase

CH3O CH3O O O

Manganese peroxidase is a hemoprotein involved in the oxidative degradation of lignin in white rot + 2 Mn(II) + 2 H + H2O2 2 Mn(III) + 2 H2O basidiomycetes. Manganese Peroxidase 20

Enzymes for Starch Hydrolysis

CH2OH CH OH 2 d O O Amylopectin is a polymer of α-(1→4)- -glycopyranosyl

OH OH units with approximately 4% α-(1→6)-d-glycopyranosyl O O branching. OH OH x O

CH2 CH2OH CH2OH CH2OH O O O O

OH OH OH OH

O O O O O OH OH OH OH

α-Amylase n

CH2OH CH2OH O O

OH OH O O OH OH O X

CH2OH CH2OH CH2OH CH2 CH2OH O O O O O

OH OH OH OH OH O OH O O O O OH OH OH OH OH n Amyloglucosidase b-Amylase a-Glucosidase

CH2OH CH2OH O O

OH OH O O OH OH O X

CH2 CH2OH CH OH Isoamylase O O 2 O OH OH O OH HO O OH OH OH n OH O Amyloglucosidase CH2 CH2OH & a-Glucosidase O O

(terminal (1–6) residues) OH OH HO O O OH OH n

Enzymatic hydrolysis of starch-based biomass 1→4- and some preparations of this enzyme • α-Glucosidase catalyzes the hydrolysis of to yield monomeric glucose is often a multi- hydrolyze 1→6- and 1→3-α-d-glucosidic terminal 1→4-linked α-d-glucose residues enzyme process and can involve α-amylase, bonds in other polysaccharides. successively from the non-reducing ends β-amylase, amyloglucosidase, isoamylase and • Isoamylase catalyzes the hydrolysis of of maltooligo- and to a lesser extent polysaccharides with release of β-d-glucose. α-glucosidase. α−(1→6)-d-glucosidic branch linkages in Most forms of the enzyme can slowly α-Amylase catalyzes the endohydrolysis amylopectin and β-limit dextrins. • hydrolyze α−(1→6)-d-glucosidic bonds. of α−(1→4)-d-glucosidic linkages in polysaccharides containing three or more a-Amylase CH OH Amylose is a polymer of α−(1→4)-linked d-glucose units. CH2OH CH2O 2 CH2OH O O O O α-(1→4)-glucopyranosyl units. • β-Amylase catalyzes the exohydrolysis OH OH OH OH O O O O O of α−(1→4)-d-glucosidic linkages in OH OH OH OH polysaccharides resulting in the successive liberation of maltose units from the non- b-Amylase n CH OH CH OH reducing ends of the chains. CH2OH 2 2 O O O • Amyloglucosidase (glucoamylase) catalyzes OH OH OH O O O the hydrolysis of terminal α−(1→4)-d- OH OH OH OH glucose residues successively from the Amyloglucosidase n non-reducing ends of maltooligo- and a-Glucosidase

polysaccharides with release of β-d-glucose. CH2OH CH2OH Most forms of the enzyme can rapidly O O OH OH hydrolyze α−(1→6)-d-glucosidic bonds OH O O when the next bond in the sequence is OH OH Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 21

▼ α-Amylase α-Amylase from Bacillus amyloliquefaciens  powder, activity: ~50 units/mg 1,4-α-d-Glucan Glucano-hydrolase One unit corresponds to the amount of enzyme α-Amylase from Aspergillus oryzae [9000‑85‑5] which liberates 1 μmole maltose per minute 1,4-α-d-Glucan-glucanohydrolase An endoamylase that randomly hydrolyzes at pH 6.9 at 25 °C (starch acc. to Zulkowsky, [9001‑19‑8] α-(1→4)-glycosidic linkages in amylose and Cat. No. 85642, as substrate).  lyophilized powder, activity: 150–250 units/mg amylopectin. The breakdown products are Applications in (selective) hydrolysis/ protein (biuret) oligosaccharides and dextrins of varying chain condensations of glycosidic bonds.1,2 Crude length. This enzyme is active at high temperatures (70−90 °C). Lit cited: 1. Y. Takasaki, Agric. Biol. Chem. 49, 1091 (1985); One unit will liberate 1.0 mg of maltose from 2. C.S. Su, C.P. Yang, J. Chem. Technol. Biotechnol. 48, 313 starch in 3 min at pH 6.9 at 20 °C. One unit is the amount of enzyme which (1990) dextrinizes 5.26 g of dry starch per hour under 10070-10G 10 g standard conditions. A6211 Inquire 10070-50G 50 g  Fungamyl® 800 L aqueous solution, activity:  BAN™ 240L liquid, activity: ≥250 units/g ≥800 FAU/g A product of Novozyme Corp. α-Amylase from barley malt 1,4-α-d-Glucan-glucanohydrolase A product of Novozyme Corp. A7595-50ML 50 mL [9000‑90‑2] A7595-250ML 250 mL A8220-50ML 50 mL  powder A8220-250ML 250 mL α-Amylase from Bacillus licheniformis β-amylase activity: ≥1 unit/mg solid  Taka-Diastase from Aspergillus oryzae; Taka-Amyl 1,4-α-d-Glucan-glucanohydrolase α-amylase activity: ≥1 unit/mg solid ase A powder, activity: ~1.5 units/mg (~0.2 U acc. [9000‑85‑5] Package size based on α-amylase activity to Willstätter) One unit will liberate 1.0 mg of maltose from One unit corresponds to the amount of enzyme starch in 3 min at pH 6.9 at 20 °C. One unit will liberate 1.0 mg of maltose from which liberates 1 μmol maltose per minute at pH starch in 3 min at pH 6.9 at 20 °C.  Termamyl® 120 saline solution, activity: 6.0 and 25 °C (starch acc. to Zulkowsky, Fluka No. A2771 Inquire 85642, as substrate). 500–1,000 units/mg protein (biuret) Aqueous solution containing approx. 15% sodium Molecular weight1; Product distribution in the α-Amylase from human pancreas chloride and 25% sucrose. hydrolysis of amylose2; Application in (selective) [9000‑90‑2] hydrolysis/condensation of glycosidic bonds3; Reported to be heat stable at temperatures as  lyophilized powder, activity: ≥100 units/mg 4 high as ~90 °C. Suitable for use in thiamine determination . protein A product of Novozyme Corp. Lit cited: 1. T. Takagi, J. Biochem. 89, 363 (1981); 2. H. Lyophilized from Tris buffer containing NaCl and Kondo et al., J. Biochem. 87, 1053 (1980); 3. K. Fujita et al., A3403-500KU 500000 units CaCl2. Bull. Chem. Soc. Jpn. 62, 3150 (1989); 4. P.W. Defibaugh, J. A3403-1MU 1000000 units Assoc. Off. Anal. Chem. 70, 514 (1987) Prepared by modified method of Levitzki et al. A3403-5MU 5000000 units One unit will liberate 1.0 mg of maltose from 86250-100G 100 g  lyophilized powder, activity: 500–1,500 units/mg starch in 3 min at pH 6.9 at 20 °C. 86250-500G 500 g protein A9972-100UG 100 μg  spray-dried (powder), activity: ≥150 units/mg Heat-stable formulation protein (biuret) Lyophilized powder containing potassium α-Amylase from human saliva One unit will liberate 1.0 mg of maltose from phosphate 1,4-α-d-Glucan-glucanohydrolase starch in 3 min at pH 6.9 at 20 °C. [9000‑90‑2] A4551-100MG 100 mg A4551-1G 1 g  lyophilized powder, activity: 300–1,500 units/mg A9857-250KU 250000 units protein A9857-1MU 1000000 units α-Amylase from Bacillus subtilis Lyophilized powder containing (NH4)2SO4 and A9857-5MU 5000000 units [9000‑90‑2] sodium citrate. α-Amylase from Bacillus sp.  powder, activity: ~380 units/mg One unit will liberate 1.0 mg of maltose from starch in 3 min at pH 6.9 at 20 °C. One unit is the amount of enzyme which liberates  ammonium sulfate suspension, activity: 1 μmole of maltose per minute at pH 6.9 and A1031-1KU 1000 units 40–90 units/mL packed gel 25 °C (using Cat. No. 85642 as substrate) A1031-5KU 5000 units Suspension in 2.0 M (NH ) SO , pH 7.0 4 2 4 Heat stability of bacterial α-amylases1; Action  lyophilized powder, activity: 1,000–3,000 units/ One unit will liberate 1.0 μmole of maltose from pattern on sweet potato starch, amylose and mg protein starch per min at pH 6.9 at 30 °C. (Each unit is amylopectin2; Action on native wheat starch.3 Lyophilized powder containing (NH4)2SO4 and equivalent to 0.7 of mg maltose liberated per 3 sodium citrate min. at 20 °C) Lit cited: 1. J.E. Anderson et al., J. Food Sci. 48, 1622 (1983); 2. P.L. Chang Rupp, S.J. Schwartz, J. Food Biochem. 12, Chromatographically purified 191 (1988); 3. P. Colonna et al., Biotechnol. Bioeng. 31, 895 A0909 Inquire One unit will liberate 1.0 mg of maltose from (1988). starch in 3 min at pH 6.9 at 20 °C. 10069-250MG 250 mg A0521-100UN 100 units 10069-1G 1 g A0521-500UN 500 units A0521-2.5KU 2500 units 22

α-Amylase from porcine pancreas One unit will liberate 1.0 mg of maltose from One unit will liberate 1.0 mg of glucose from Molecular mass: 51–54 kDa. starch in 3 min at pH 4.8 at 20 °C. starch in 3 min at pH 4.5 at 55 °C. α-Amylase isolated from porcine pancreas is a A7130-10KU 10000 units A7420-5MG 5 mg glycoprotein. It is a single polypeptide chain of A7130-50KU 50000 units A7420-25MG 25 mg ~475 residues containing two SH groups and A7130-250KU 250000 units A7420-100MG 100 mg four disulfide bridges and a tightly bound Ca2+ β-Amylase from sweet potato  powder, activity: ~120 units/mg necessary for stability. Chloride ions are necessary One unit corresponds to the amount of enzyme for activity and stability. The pH range for activity is  ammonium sulfate suspension, activity: which liberates 1 μmol glucose per minute at 5.5 to 8.0, with the pH optimum at 7. 1% ≥750 units/mg protein (E /280) pH 4.8 and 60 °C (starch, Fluka No. 85642, as One unit will liberate 1.0 mg of maltose from Crystalline suspension in 2.3 M (NH4)2SO4 substrate). starch in 3 min at pH 6.9 at 20 °C. One unit will liberate 1.0 mg of maltose from Synthesis of hetero-oligosaccharides by  activity: ≥10 units/mg solid starch in 3 min at pH 4.8 at 20 °C. glucoamylase in reverse1. Contains lactose View more information on enzymes for complex Lit cited: 1. R.A. Rastall et al., Biotechnol. Lett. 13, 501 (1991) Package size based on α-amylase activity carbohydrate analysis at sigma-aldrich.com/ 10113-1G 1 g A3176-500KU 500000 units enzymeexplorer 10113-5G 5 g A3176-1MU 1000000 units A7005-10KU 10000 units A3176-2.5MU 2500000 units A7005-25KU 25000 units Amyloglucosidase from Rhizopus sp. A3176-5MU 5000000 units A7005-50KU 50000 units  activity: ≥5,000 units/g solid A3176-10MU 10000000 units A7005-100KU 100000 units One unit will liberate 1.0 mg of glucose from  DFP Treated, saline suspension, activity: β-Amylase ▲ starch in 3 min at pH 4.5 at 55 °C. 1% 700–1400 units/mg protein (E /280) Using soluble starch, almost theoretical yields of Suspension in 2.9 M NaCl solution containing α-Amylase, heat-stable glucose are obtained. α-Amylase from Bacillus licheniformis; 1,4-α-D-Glucan- 3 mM CaCl2 glucanohydrolase DFP treated. 2× crystallized A7255 Inquire [9000‑85‑5] View more information on enzymes for complex ▲  solution, for use in Total Dietary Fiber Assay, TDF- Amyloglucosidase carbohydrate analysis at sigma-aldrich.com/ 100A enzymeexplorer ▼ α-Glucosidase A3306-10ML 10 mL A6255-10MG 10 mg α-d-Glucoside glucohydrolase [9001‑42‑7] A6255-25MG 25 mg Amylase, Maltogenic from Bacillus sp. Protein determined by biuret. A6255-100MG 100 mg Maltogenic Amylase; Glucan 1,4-α-maltohydrolase  DFP treated, ammonium sulfate suspension, Novamyl 1000BG α-Glucosidase from Bacillus 1% activity: ≥500 units/mg protein (E /280) A2986-10G 10 g stearothermophilus Suspension in 3.2 M (NH ) SO , pH 6.1 4 2 4 ▼ Amyloglucosidase Maltase A2643 Inquire Exo-1,4-α-glucosidase; 1,4-α-d-Glucan glucohydrolase;  Maltase lyophilized powder, activity: Glucoamylase ≥50 units/mg protein  PMSF treated, saline suspension, activity: [9032‑08‑0] 1% Lyophilized powder containing potassium 700–1400 units/mg protein (E /280) phosphate buffer salt Suspension in 2.9 M NaCl solution containing Amyloglucosidase from Aspergillus niger One unit will liberate 1.0 μmole of d-glucose from 3 mM CaCl2.  lyophilized, powder, activity: ~70 units/mg p-nitrophenyl α-d-glucoside per min at pH 6.8 at 2× crystallized For glycogen determination in whole yeast 37 °C. A4268-25MG 25 mg cells; Synthesis of hetero-oligosaccharides by Activity using maltose as substrate at pH 6.0 at A4268-100MG 100 mg glucoamylase in reverse. 25 deg C is ~2X greater than that obtained using α-Amylase ▲ One unit corresponds to the amount of enzyme p-nitrophenyl-α-d-glucoside as substrate at pH 6.8 which liberates 1 μmole of glucose per minute at at 37 °C. ▼ β-Amylase pH 4.8 and 60 °C (starch acc. to Zulkowsky, G3651-250UN 250 units Cat. No. 85642, as substrate). 1,4-α-d-Glucan maltohydrolase [9000‑91‑3] 10115-1G-F 1 g α-Glucosidase from rice 10115-5G-F 5 g Maltase β-Amylase from barley  lyophilized powder, activity: 30–60 units/mg  ammonium sulfate suspension, activity:  activity: 20–80 units/mg protein (biuret) protein (biuret) 40–80 units/mg protein Crude Lyophilized powder containing less than 0.02% Suspension in 2.8 M (NH ) SO solution glucose 4 2 4 One unit will convert 1.0 μmole of maltose to 2.0 μmoles of d-glucose per min at pH 4.0 at 37 °C.

G9259-100UN 100 units Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 23

α-Glucosidase from Saccharomyces View more information on enzymes for complex Fuel Ethanol Residual Saccharides Mix cerevisiae carbohydrate analysis at sigma-aldrich.com/ Quantitative standard for monitoring unfermented enzymeexplorer α-d-Glucosidase; Maltase from yeast Hydrolysis of sugars present during the process of transforming terminal, non-reducing 1→4-linked d-glucose G5003-100UN 100 units corn feedstock into ethanol. residues with release of d-glucose. G5003-1KU 1000 units Components Glycerol 1 % (w/v) For the determination of α-amylase and the α-Glucosidase ▲ D-(+)-Glucose 2 % (w/v)

synthesis of various 1’-O-sucrose and 1-O-fructose Maltotriose (DP3) 1 % (w/v) esters Maltose monohydrate 2 % (w/v) Ethanol Analysis Reagents L-(+)-Lactic acid .3 % (w/v)  recombinant, expressed in unspecified host, Acetic acid .3 % (w/v) lyophilized powder, activity: ≥125 units/mg ASTM™ D5501 Denatured Fuel Ethanol Dextrin 3.25 % (w/v) protein Ethanol 12 % (w/v) Standards Kit Lyophilized powder containing potassium phosphate buffer salt pH 7.15 and approx. 70% Use this quantitative calibration standard kit to 48468-U 10 × 2 mL lactose determine if ethanol and gasoline fuel blends comply with federal and state laws. A certificate of Ethanol standards 10% (v/v) One unit will liberate 1.0 μmole of d-glucose from analysis accompanies each kit. p-nitrophenyl α-d-glucoside per min at pH 6.8 at Ethanol solution; Ethyl alcohol CH3CH2OH

37 °C. suitable for D5501 per ASTM [64‑17‑5] C2H5OH FW 46.07 G0660-750UN 750 units Components  aqueous solution  lyophilized powder, activity: ≥10 units/mg protein Sol.#1 Ethanol:Heptane: Methanol (92%, 7.40%, 0.60%) Flame-sealed flint ampule Sol.#2 Ethanol:Heptane: Methanol (93%, 6.50%, 0.50%) (using p-nitrophenyl α-d-glucoside as substrate.) E2385-10AMP 10 amp Sol.#3 Ethanol:Heptane: Methanol (94%, 5.60%, 0.40%) Sold on basis of p-nitrophenyl α-d-glucoside units. Sol.#4 Ethanol:Heptane: Methanol (95%, 4.70%, 0.30%) One unit will liberate 1.0 μmole of d-glucose from Sol.#5 Ethanol:Heptane: Methanol (96%, 3.80%, 0.20%) p-nitrophenyl α-d-glucoside per min at pH 6.8 at Sol.#6 Ethanol:Heptane: Methanol (97%, 2.90%, 0.10%) 37 °C. Sol.#7 Ethanol:Heptane: Methanol (98%, 1.95%, 0.05%) 40361-U 1 pkg

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Enzymes and Reagents for BioDiesel Research Lipases for Transesterification

Enzymes and Reagents for BioDiesel Research

Lipases for Transesterification  activity: ≥10,000 U/g, recombinant, expressed in Phospholipase C from Clostridium Aspergillus niger perfringens (C. welchii) Amano Lipase PS A product of Novozyme Corp. One unit will liberate 1.0 μmole of water  (immobilized on diatomite) L4777-3G 3 g soluble organic phosphorus from egg yolk l-α- 708011-10G 10 g L4777-10G 10 g phosphatidylcholine per min at pH 7.3 at 37 °C. Amano Lipase PS, from Phospholipase C for  lyophilized powder, activity: 10–50 units/mg Burkholderia cepacia protein  activity: ≥30,000 U/g, pH 7.0, 50 °C (Optimum pH Degumming Oils P7633-25UN 25 units and temperature) P7633-125UN 125 units ▼ Phospholipase C 534641-10G 10 g P7633-500UN 500 units Phosphatidylcholine cholinephosphohydrolase; 534641-50G 50 g  Lecithinase C; Lipophosphodiesterase I; PC-PLC lyophilized powder, activity: ≥150 units/mg [9001‑86‑9] protein Amano Lipase PS-C II (immobilized Lyophilized powder in buffered salts on ceramic) Hydrolyzes the phosphate bond on phosphatidylcholine and other Chromatographically purified One unit will produce 1.0 micromole of glycerophospholipids yielding diacylglycerol; this 1-Phenethyl alcohol to 1-Phenethyl acetate per enzyme will also hydrolyze the phosphate bonds P4039-125UN 125 units min. at 25 °C in the presence of vinyl acetate of sphingomyelin, cardiolipin, choline plasmalogen P4039-500UN 500 units 534889 Inquire and ceramide phospholipids. Phospholipase C ▲ Amano lipase PS-IM (immobilized on Phospholipase C from Bacillus cereus diatomaceous earth)  activity: ≥200 units/mg protein 709603-10G 10 g Lyophilized powder containing approx. 10% protein. Remainder: trehalose, zinc sulfate, and Amano Lipase PS-C I (immobilized on potassium phosphate ceramic) One unit will liberate 1.0 μmole of water soluble One unit will produce 1.0 micromole of organic phosphorus from L-α-phosphatidylcholine 1-Phenethyl alcohol to 1-Phenethyl acetate per per min at pH 7.3 at 37 °C. min. at 25 °C in the presence of vinyl acetate P6621-250UN 250 units 534897 Inquire

Lipase from Candida sp. O O HC O HC OH RO  Novozymes® CALB L recombinant, expressed in O Lipase O HC O + 3 ROH HC OH + RO Aspergillus niger O O HC O HC OH RO aqueous solution Minimum 5,000 LU/G of liquid Transesterification of triglycerides utilizing commercial lipase preparations is currently under investigation as a low L3170-50ML 50 mL energy input alternative to current “chemical” methods.

Lipase acrylic resin from Candida O antarctica CH3 O O P OH HC O Phospholipase C H3C N Novozym® 435 O OH CH3 + [9001‑62‑1] HC O O CH3 O O P CH2 HC O Immobilized preparation of a thermostable lipase, H C 3 N +H2O O particularly useful in the synthesis of esters and OH CH3 HC O amides, and has a broad substrate specificity. HO CH2 Lipase from (B lipase) Candida antarctica produced by submerged fermentation of a genetically Degumming of feedstock oils by hydrolyisis of phospholipids can be catalyzed by phospholipase C. Treatment of modified Aspergillus oryzae microorganism and phosphatidylethanol, phosphatidylcholine and phosphatidylinositol with phospholipase C enables the hydrolysis adsorbed on a macroporous resin. and phase separation of the hydrophilic phosphate-containing contaminants. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 25

BioDiesel Analysis GC capillary columns BioDiesel Calibration Standards Temp. Limits: GC Columns for BioDiesel Analysis Supelco glycerin impurities in biodiesel • –60 °C to 380 °C (isothermal) reference solutions meet the specifications MET-Biodiesel • –60 °C to 430 °C (programmable) called out in ASTM D6584 and DIN EN 14105. This rugged metal column was designed Our reference solutions have been carefully 1. Glycerin 4 specifically for the determination of free and total 2. Butanetriol (I.S.) 3. Monoolein formulated to save 4–6 hours in prep time, glycerin in B100 biodiesel samples. Features and 4. Tricaprin (I.S.) 5. Diolein minimize the possibility of human error, benefits of this column include: 6. Triolein 2 and eliminate the need to prepare pyridine • Metal is used as the column material, virtually eliminating accidental column dilutions. Our standards offer: 3 5 breakage during handling 1 6 • Raw materials tested for identity and purity

01020 • Metal columns do not require a coating Min • Stock solutions for preparing your own to provide strength (NOTE: the protective multi-component solutions polyimide coating on the outside of fused 28668-U 1 ea Multilevel, multi-component solutions silica columns slowly burns off at 380 °C, • Derivatization ready for derivatization exposing bare fused silica which is fragile and susceptible to breakage) N-Methyl-N-(trimethylsilyl)trifluoroacet- • Monoglyceride commercial mixture for EN14105 • Includes a guard that both protects the amide analytical column from excess reagent MSTFA; N-Trimethylsilyl-N-methyl tri O Internal standard solutions CH3 • fluoroacetamide and non-volatile compounds, extending F3C N Si CH3 [24589‑78‑4] CF CON(CH )Si(CH ) H3C CH3 Certificate of composition shipped with column life, and acts as a retention gap, 3 3 3 3 • FW 199.25 each standard minimizing peak broadening Silylating agent used to form volatile derivatives 2 • Instructions for derivatization with each kit • The guard is an integrated guard, thereby for GC-MS analysis. providing protection with a leak-free Improved silylation procedure for Our standards are manufactured under connection (the guard and analytical simultaneous detection of steroid hormones the Quality Management System which is 1 column are one continuous piece of 17-α-ethynylestradiol and estrone. registered under ISO 9001:2000. tubing; there is no union between the Lit cited: 1. J. Chromatogr. 1108, 121 (2006); 2. Phosphorus, guard and analytical column) Sulfur Silicon Relat. Elem. 88, 53 (1994). 394866-10X1ML 10 × 1 mL • Low bleed characteristic, even at 380 °C 394866-5ML 5 mL • Provides good peak shape and resolution 394866-25ML 25 mL for all glyceride impurities of interest • Able to separate glycerin in addition to mono-/diglycerides (as methyl esters) plus triglycerides from the FAMEs • A maximum temperature of 380 °C (isothermal) and 430 °C (programmed) exceeds the temperature limitations specified in biodiesel methods such as ASTM D6584 and EN 14105 26

Biodiesel Calibration Standards

Name Suitability Composition Cat. No. ASTM® D6584 Individual Standard Solution and Internal D6584 ASTM ASTM® D6584 Standard Solution 1 (Supelco 44899-U), 1 mL 44918-U Standards Kit ASTM® D6584 Standard Solution 2 (Supelco 44914-U), 1 mL ASTM® D6584 Standard Solution 3 (Supelco 44915-U), 1 mL ASTM® D6584 Standard Solution 4 (Supelco 44916-U), 1 mL ASTM® D6584 Standard Solution 5 (Supelco 44917-U), 1 mL ASTM D6584 1,2,4-Butanetriol Solution, Internal Standard #1 (Supelco 44896-U), 5 mL ASTM D6584 Tricaprin Solution, Internal Standard #2 (Supelco 44897-U), 5 mL ASTM® D6584 Standard Solution 1 D6584 ASTM 1,3-Diolein, 50 μg/mL 44899-U Glycerol, 5 μg/mL Monoolein, 100 μg/mL Glyceryl trioleate, 50 μg/mL ASTM® D6584 Standard Solution 2 D6584 ASTM 1,3-Diolein, 100 μg/mL 44914-U Glycerol, 15 μg/mL Monoolein, 250 μg/mL Glyceryl trioleate, 100 μg/mL ASTM® D6584 Standard Solution 3 D6584 ASTM 1,3-Diolein, 200 μg/mL 44915-U Glycerol, 25 μg/mL Monoolein, 500 μg/mL Glyceryl trioleate, 200 μg/mL ASTM® D6584 Standard Solution 4 D6584 ASTM 1,3-Diolein, 350 μg/mL 44916-U Glycerol, 35 μg/mL Monoolein, 750 μg/mL Glyceryl trioleate, 350 μg/mL ASTM® D6584 Standard Solution 5 D6584 ASTM 1,3-Diolein, 500 μg/mL 44917-U Glycerol, 50 μg/mL Monoolein, 1000 μg/mL Glyceryl trioleate, 500 μg/mL EN 14105:2003 Monoglyceride Stock Solution EN 14105 DIN Monoolein 49446-U Monopalmitin Monostearin EN 14105:2003 Standard Solution 1 EN 14105 DIN Butanetriol, 80 μg/mL 49441-U 1,3-Diolein, 50 μg/mL Glycerol, 5 μg/mL Monoolein, 250 μg/mL Tricaprin, 800 μg/mL Triolein, 50 μg/mL EN 14105:2003 Standard Solution 2 EN 14105 DIN Butanetriol, 80 μg/mL 49442-U Diolein, 200 μg/mL Glycerol, 20 μg/mL Monoolein, 600 μg/mL Tricaprin, 800 μg/mL Triolein, 150 μg/mL EN 14105:2003 Standard Solution 3 EN 14105 DIN Butanetriol, 80 μg/mL 49443-U Diolein, 350 μg/mL Glycerol, 35 μg/mL Monoolein, 950 μg/mL Tricaprin, 800 μg/mL Triolein, 300 μg/mL EN 14105:2003 Standard Solution 4 EN 14105 DIN Butanetriol, 80 μg/mL 49444-U Diolein, 500 μg/mL Glycerol, 50 μg/mL Monoolein, 1250 μg/mL Tricaprin, 800 μg/mL Triolein, 400 μg/mL EN 14105:2003 Standard Solution Kit EN 14105 DIN EN 14105:2003 Standard Solution 1 (Supelco 49441-U), 1 mL 49445-U EN 14105:2003 Standard Solution 2 (Supelco 49442-U), 1 mL EN 14105:2003 Standard Solution 3 (Supelco 49443-U), 1 mL EN 14105:2003 Standard Solution 4 (Supelco 49444-U), 1 mL Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 27

Karl Fischer Titration—HYDRANAL® the storage tanks. Water content can be One-Component Volumetric Reagents Reagents for the Determination of Water determined accurately and reproducibly by Biodiesel contains components of different in Biodiesel Karl Fischer Titration (volumetric, coulometric, chain lengths. Therefore, a solubilizer is The presence of water in biodiesel poses and KF-oven methods) using reliable, often required to dissolve or disperse it in problems for a number of reasons. Some superior, and proven HYDRANAL reagents order to extract the moisture. A number of the water is residual from processing (EN 14214). of HYDRANAL media are available for while some comes from condensation in effective dispersion of biodiesel. HYDRANAL- Composite 2 is used as the titration agent.

One Component Reagents Name Grade Cat. No. HYDRANAL®-Composite 1 one-component reagent for volumetric Karl Fischer titration 34827-500ML-R 34827-6X500ML-R 34827-1L-R 34827-6X1L-R HYDRANAL®-Composite 2 one-component reagent for volumetric Karl Fischer titration 34806-500ML 34806-6X500ML 34806-1L 34806-6X1L 34806-2.5L 34806-4X2.5L HYDRANAL®-Composite 5 one-component reagent for volumetric Karl Fischer titration 34805-500ML-R 34805-6X500ML-R 34805-1L-R 34805-6X1L-R 34805-2.5L-R 34805-4X2.5L-R HYDRANAL®-Composite 5 K one-component reagent for volumetric Karl Fischer titration in ketones and aldehydes 34816-500ML-R 34816-6X500ML-R 34816-1L-R 34816-6X1L-R 34816-2.5L-R 34816-4X2.5L-R HYDRANAL®-CompoSolver E – 34734-1L-R 34734-6X1L-R 34734-2.5L-R 34734-4X2.5L-R HYDRANAL®-KetoSolver – 34738-500ML-R 34738-6X500ML-R 34738-1L-R 34738-6X1L-R HYDRANAL®-LipoSolver CM – 37855-1L 37855-6X1L HYDRANAL®-LipoSolver MH – 37856-1L 37856-6X1L HYDRANAL®-Medium K – 34698-1L-R 34698-6X1L-R HYDRANAL®-Methanol dry – 34741-1L-R 34741-6X1L-R 34741-2.5L-R 34741-4X2.5L-R HYDRANAL®-Methanol Rapid – 37817-1L-R 37817-6X1L-R 37817-2.5L-R 37817-4X2.5L-R HYDRANAL®-Solver (Crude) oil – 34697-1L-R 34697-6X1L-R 34697-2.5L-R 34697-4X2.5L-R HYDRANAL®-Working Medium K – 34817-1L 34817-6X1L Karl-Fischer reagent for titrimetric determination of water with one solution 36115-1L 36115-6X1L 28

Two-Component Volumetric Reagents

Name Grade Cat. No. HYDRANAL®-Solvent – 34800-1L-R 34800-6X1L-R 34800-2.5L-R 34800-4X2.5L-R HYDRANAL®-Solvent E – 34730-500ML-R 34730-6X500ML-R 34730-1L-R 34730-6X1L-R 34730-2.5L-R 34730-4X2.5L-R HYDRANAL®-Solvent CM – 34812-1L 34812-6X1L 34812-2.5L 34812-4X2.5L HYDRANAL®-Solvent Oil – 34749-1L 34749-6X1L HYDRANAL®-Titrant 2 – 34811-500ML 34811-6X500ML 34811-1L 34811-6X1L HYDRANAL®-Titrant 5 – 34801-500ML 34801-6X500ML 34801-1L 34801-6X1L 34801-2.5L 34801-4X2.5L HYDRANAL®-Titrant 2 E – 34723-1L-R 34723-6X1L-R HYDRANAL®-Titrant 5 E – 34732-100ML-R 34732-500ML-R 34732-6X500ML-R 34732-1L-R 34732-6X1L-R 34732-2.5L-R 34732-4X2.5L-R Karl-Fischer reagent for titrimetric determination of water with two separate solutions 36116-1L 36116-6X1L Karl-Fischer reagent for titrimetric determination of water with two separate solutions 36117-1L 36117-6X1L Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 29

Coulometric Titration Reagents The coulometric procedure is more sensitive than the volumetric titration method. Determination of water is carried out in a coulometric cell (with or without diaphragm). The precision of the coulometric cell and the sample manipulation can be tested by means of HYDRANAL Water Standard 0.10.

Name Grade Cat. No. HYDRANAL®-Coulomat A anolyte for coulometric Karl Fischer titration (for cells with diaphragm) 34807-500ML 34807-6X500ML HYDRANAL®-Coulomat E – 34726-500ML-R 34726-6X500ML-R HYDRANAL®-Coulomat AD reagent for coulometric Karl Fischer titration for cells without diaphragm 34810-500ML-R 34810-6X500ML-R HYDRANAL®-Coulomat AF 7 anolyte for coulometric Karl Fischer titration (suitable for coulometer AF7) 34829-1L-R 34829-6X1L-R HYDRANAL®-Coulomat AG anolyte for coulometric Karl Fischer titration. Suitable for cells with and without diaphragm 34836-500ML-R 34836-6X500ML-R 34836-1L-R 34836-6X1L-R HYDRANAL®-Coulomat AG-H anolyte for coulometric Karl Fischer titration in long-chained hydrocarbons 34843-500ML (free of halogenated hydrocarbons) 34843-6X500ML HYDRANAL®-Coulomat AG-Oven – 34739-500ML-R 34739-6X500ML-R HYDRANAL®-Coulomat AK anolyte for coulometric Karl Fischer titration in ketones (for cells with and without diaphragm) 34820-500ML-R 34820-6X500ML-R HYDRANAL®-Coulomat CG catholyte for coulometric Karl Fischer titration. Free of halogenated hydrocarbons 34840-50ML-R 34840-6X50ML-R HYDRANAL®-Coulomat CG-K – 34821-50ML-R 34821-6X50ML-R HYDRANAL®-Coulomat Oil – 34868-100ML-R 34868-6X100ML-R 34868-500ML-R 34868-6X500ML-R

Karl Fischer Oven products This method is suitable for samples that release water at a higher temperature. In this method, HYDRANAL-Coulomat CG is placed in the cathode chamber and HYDRANAL Coulomat AG Oven is added to the anode chamber of a coulometric cell with diaphragm. A cell without a diaphragm requires only the Coulomat AG Oven. HYDRANAL Coulomat AG-H or HYDRANAL Coulomat AD may be used as a replacement for HYDRANAL Coulomat AG Oven. HYDRANAL Molecular Sieve 0.3 mm is a drying medium for the carrier gas.

Name Grade Cat. No. HYDRANAL®-Coulomat AG-Oven – 34739-500ML-R 34739-6X500ML-R HYDRANAL®-Coulomat CG catholyte for coulometric Karl Fischer titration. Free of halogenated hydrocarbons 34840-50ML-R 34840-6X50ML-R HYDRANAL®-Molecular sieve 0.3 nm – 34241-250G-R 34241-1KG-R HYDRANAL®-Water Standard KF-Oven, – 34693-10G-R 140–160 °C HYDRANAL®-Water Standard KF-Oven – 34748-10G-R 220 °C–230 °C 34748-6X10G-R 30

Metabolite Standards for Mevalonate and Isoprenoid Pathway Analysis

Name Cat. No. ETHANOL ISOPENTANOL PROPANOL BUTANOL Acetoacetyl coenzyme A sodium salt hydrate A1625-5MG A1625-10MG A1625-25MG Acetaldehyde Valeryl-CoA Propionyl-CoA Butyryl-CoA Acetyl coenzyme A sodium salt, powder A2056-1MG TCA-Cycle A2056-5MG Esters A2056-10MG G3P PEP Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetyl coenzyme A trilithium salt A2181-1MG A2181-5MG Even- numbered A2181-10MG fatty acid Butyryl coenzyme A lithium salt hydrate B1508-5MG 1-Deoxy-D-xylulose- 3-Hydroxy-3-methyl- Odd-numbered 5-phosphate glutaryl-CoA fatty acid B1508-10MG B1508-25MG Esters 1-Deoxy-d-xylulose-5-phosphate sodium salt 13368-1MG 2-C-Methyl-D- Mevalonate Even-numbered fatty alcohol 13368-5MG erythritol-4-phosphate Odd-numbered fatty alcohol γ,γ-Dimethylallyl pyrophosphate triammonium salt D4287-1VL Mevalonate-5-phosphate D4287-5VL Isopentanol Farnesyl monophosphate ammonium salt solution, F1803-5VL Even-numbered alkane methanol:ammonia solution Mevalonate-5-pyrophosphate Isopentenol Farnesyl pyrophosphate ammonium salt solution, F6892-1VL Odd-numbered methanol:ammonia solution F6892-5VL alkane Dimethylallyl-pyrophosphate Isopentenyl-pyrophosphate Geranylgeranyl monophosphate ammonium salt solution, G2543-5VL methanol:ammonia solution Geranylgeranyl pyrophosphate ammonium salt solution G6025-1VL Geranyl-pyrophosphate methanol:ammonia solution G6025-5VL Geranyl monophosphate ammonium salt solution, G2293-1VL (E,E)-Farnesyl-pyrophosphate Monoterpene Geraniol methanol:ammonia solution G2293-5VL Geranyl pyrophosphate ammonium salt G6772-1VL Geranylgeranyl-pyrophosphate G6772-5VL d l -Glyceraldehyde 3-phosphate solution G5251-25MG Sesquiterpene Farnesol G5251-100MG Diterpene Geranylgeraniol d l -3-Hydroxy-3-methylglutaryl coenzyme A sodium salt H6132-5MG H6132-10MG Alternatives to the classical glycolysis and fermentation pathways are also being studied as Isopentenyl monophosphate ammonium salt solution I1157 sources of common biofuels as well as potential sources of new biofuels such as isoprenoids. Isopentenyl pyrophosphate triammonium salt solution I0503-1VL The mevalonate-isoprenoid pathway may prove to be a rich source of a variety of hydrocar- I0503-5VL bon-based biofuels. (R)-Mevalonic acid sodium salt 41288 Sigma-Aldrich’s comprehensive and unique range of metabolites enables new approaches (±)-Mevalonic acid 5-phosphate trilithium salt hydrate 79849-10MG towards molecular understanding of the relevant mevalonate pathway (center) and the DXP 79849-50MG pathway (left) towards the isoprenoid compounds (bottom), the fatty-acid or fatty acyl-CoA (R)-Mevalonic acid 5-pyrophosphate tetralithium salt 77631 pathways (top and right) towards the different alcohols, esters and alkanes. (±)-Mevalonic acid 5-pyrophosphate tetralithium salt 94259-10MG 94259-50MG (R)-(−)-Mevalonolactone 68519-100MG (±)-Mevalonolactone M4667-1G Phospho(enol)pyruvic acid monopotassium salt P7127-100MG Phospho(enol)pyruvic acid monosodium salt hydrate P0564-100MG Phospho(enol)pyruvic acid trisodium salt hydrate P7002-100MG n-Propionyl coenzyme A lithium salt P5397-5MG Pyruvic acid 107360-25G Sodium pyruvate 15990-25G Renewable and Alternative Energy Web Portal

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