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lab carbohydrate metabolism BY SOHAIL SARWAR from University of LHH

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lab carbohydrate metabolism BY SOHAIL SARWAR from University of LHH

  1. 1. L.A.B.: Carbohydrate Metabolism Transport of sugars across cell membrane Catabolism of sugars for energy Synthesis of polysaccharides Homolactic fermentation Mixed acid fermentation Heterolactic fermentation <ul><li>Pathways for glucose fermentation </li></ul><ul><li>Pathways for other hexoses </li></ul><ul><li>Pathways for pentoses </li></ul><ul><li>Pathway for citrate </li></ul><ul><li>Disaccharides </li></ul>
  2. 2. Selected references <ul><li>Salminen, S. and A. vonWright. 1998. Lactic acid bacteria . Marcel Dekker, Inc., New York, NY. pp. 51-56 (sugar transport), pp. 22-43 (degradation of sugars for energy). </li></ul><ul><li>Marth, E.H. and J.L. Steele (eds.). 1998. Applied Dairy Microbiology . Marcel Dekker, Inc., New York, NY. Pp. 178-187. </li></ul>
  3. 3. Sugar Transport in L.A.B. cell membrane OUT IN Concentration Gradient (  S 1 &  S 2 ) PEP Energy from: Sugar Sugar H + H + Symport Permease Sugar Sugar- PO 4 PEP-PTS Antiport Sugar 1 Sugar 1 Sugar 2 Sugar 2 Permease PEP Pyruvate EIIBC S EIIA S HPr EI ATP H + H + ADP F 1 F 0 ATPase PMF (  pH +  )
  4. 4. PEP-PTS
  5. 5. Sugar Transport Systems are Sugar-Specific cell membrane OUT IN Lactose Lactose H + H + Lactose Permease Glucose Glucose H + H + Glucose Permease Galactose Galactose H + H + Galactose Permease Also sugar specific…… Antiport permease PEP-PTS – EIIA and EIIBC
  6. 6. Example: Lactose Transport Type of Transport System for a Sugar is Species Specific PEP-PTS: Lactococcus Lactobacillus casei Antiport (lactose/galactose): S. thermophilus Lactobacillus delbrueckii subsp. bulgaricus Symport Lactobacillus helveticus Lactobacillus acidophilus Leuconostoc Pediococcus Lactose Lactose H + H + Lactose Lactose Galactose Galactose Lactose Lactose-PO 4
  7. 7. Homolactic Fermentation of Glucose (Embden-Meyerhof Pathway) Glucose Pyruvate = key intermediate (Glycolysis + LDH) Fructose-1,6-diphosphate ATP ADP (2) 3-Phosphoglycerate (2) ATP (2) ADP (2) 1,3-Diphosphoglycerate (2) H 2 PO 4 - (2) NAD + (2) NADH + (2) H + (2) 2-Phosphoglycerate (2) Phosphoenolpyruvate ( PEP ) (2) H 2 O (2) Pyruvate (2) ATP (2) ADP (2) Glyceraldehyde-3-phosphate Dihydroxyacetone-phosphate FDP aldolase Products : 2 ATP 2 Lactate Key enzymes : FDP aldolase Lactate dehydrogenase (2) Lactate (2) NAD + (2) NADH + (2) H + Lactate dehydrogenase (LDH) cofactor regeneration O-P-O O O phosphate group Glucose-6-phosphate ATP ADP O-P-O O O Fructose-6-phosphate
  8. 8. Two roles for PEP Transport (PEP-PTS) or ATP generation
  9. 9. Mixed Acid Fermentation: Alternative endproducts for pyruvate Glucose (2) ATP (2) ADP (2) H 2 PO 4 - (2) NAD + (2) NADH + (2) H + (2) Pyruvate (2) ATP (2) ADP 2 ATP 2 ADP (2) Glyceraldehyde-3-phosphate Dihydroxyacetone-phosphate FDP aldolase (2) Lactate (2) NAD + (2) NADH + (2) H + (LDH) Homolactic cofactor regeneration ATP ADP Acetate Acetyl-phosphate (2) Acetyl-CoA Acetaldehyde Ethanol NAD + NADH + H + (2) Formate (2) CoA NAD + NADH + H + CoA H 2 PO 4 - CoA Products : 3 ATP 2 Formate 1 Ethanol 1 Acetate Key enzymes : FDP aldolase Pyruvate formate lyase (PFL)
  10. 10. Homolactic vs. Mixed Acid Fermentation Homolactic fermentation prevails when glucose is abundant. Mixed acid fermentation prevails when sugars are limited – “semi-starvation”. Substrate availability and the nature of the substrate determine which pathway is used….. Mixed acid fermentation prevails during growth on galactose as the primary sugar source. Homolactic fermentation prevails under aerobic conditions because the pyruvate formate lyase (PFL) enzyme is oxygen sensitive.
  11. 11. Glucose Heterolactic Fermentation of Glucose (Pentose Phosphate Pathway) O 2 --- Aerobic conditions Glucose-6-phosphate ATP ADP 1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate ( PEP ) Pyruvate Lactate ATP ADP H 2 PO 4 - NAD + NADH + H + (2) H 2 O ATP ADP NAD + NADH + H + LDH 6-phospho-gluconate NAD + NADH + H + Xylulose-5-phosphate Ribulose-5-phosphate NAD + NADH + H + CO 2 Products : 2 ATP 1 CO 2 1 Lactate 1 Acetate Key enzymes : Phosphoketolase Lactate dehydrogenase NADH oxidase Glyceraldehyde-3-phosphate H 2 PO 4 - Acetyl-phosphate Phosphoketolase ATP ADP Acetate NAD + NADH + H + NAD + NADH + H + H 2 O 2 2 H 2 O NADH oxidase NADH oxidase
  12. 12. Glucose Glucose-6-phosphate 6-phospho-gluconate Ribulose-5-phosphate Glyceraldehyde-3-phosphate 1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate ( PEP ) Pyruvate Lactate ATP ADP ATP ADP NAD + NADH + H + (2) H 2 O ATP ADP NAD + NADH + H + LDH Heterolactic Fermentation of Glucose --- Anaerobic conditions Xylulose-5-phosphate H 2 PO 4 - CO 2 Acetyl-phosphate Phosphoketolase H 2 PO 4 - NAD + NADH + H + NAD + NADH + H + Acetyl-CoA Acetaldehyde Ethanol NAD + NADH + H + H 2 PO 4 - CoA NAD + NADH + H + CoA Products : 1 ATP 1 CO 2 1 Lactate 1 Ethanol Key enzymes : Phosphoketolase Lactate dehydrogenase
  13. 13. Differentiation of L.A.B. Genera
  14. 14. Hexoses other than glucose Fructose, mannose and galactose enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization and phosphorylation steps (when galactose is transported by permease – Leloir – next slide) Glucose Glucose-6-phosphate Fructose-6-phosphate HOMOLACTIC & MIXED ACID Glucose Glucose-6-phosphate 6-phospho-gluconate HETEROLACTIC Galactose Galactose-1-P Glucose-1-P Fructose
  15. 15. Galactose metabolism pathway depends on transport system used Galactose H + H + Galactose Permease Galactose Galactose Galactose-6-PO 4 PEP Pyruvate EI EI Homolactic or *mixed acid pathway * Mixed acid fermentation dominates if galactose is the most abundant sugar available Leloir Pathway Galactose-1-PO 4 Glucose-1-PO 4 Glucose-6-PO 4 ATP ADP Homolactic, *mixed acid, or heterolactic pathway Tagatose-1,6-diPO 4 ATP ADP (2) Glyceraldehyde- 3-phosphate Dihydroxyacetone- phosphate Tagatose Pathway Tagatose-6-PO 4
  16. 16. Some L.A.B. cannot metabolize galactose Lactose Lactose Galactose Galactose Permease cell membrane OUT IN Example: S. thermophilus and Lb. delbrueckii subsp. bulgaricus Galactose is exported via antiport system Glucose Homolactic, mixed acid or heterolactic pathway
  17. 17. Disaccharides: broken into monosaccharides before metabolized sucrose maltose lactose galactose glucose glucose fructose glucose glucose
  18. 18. Lactose Breakdown: depends on transport system + +  -galactosidase Phospho-  -galactosidase Homolactic, mixed acid, or heterolactic pathway Homolactic, mixed acid, or heterolactic pathway Tagatose pathway Leloir pathway PO 4 PO 4
  19. 19. Summary of Lactose Metabolism in L.A.B. How many ATPs from one lactose? Lactococcus Lb. casei *S. thermophilus, *Lb. delbrueckii, Lb. Helveticus, Lb. lactis * S. thermophilus, and Lb. delbrueckii do not metabolize the galactose part of lactose. They export galactose from the cell. Leuconostoc Group III Lactobacillus (Figure from Fox et al. 1990. Critical Reviews in Food Science and Nutrition. 29:237-253.) Tagatose Pathway Homolactic Pathway Leloir Pathway Heterolactic Pathway  -galactosidase phospho-  -galactosidase CO 2
  20. 20. Pentoses Pentose Pentose Pentose-PO 4 Xylulose-PO 4 or Ribulose-PO 4 Heterolactic fermentation pathway ATP ADP isomerization Pentoses cannot enter the homolactic or mixed acid pathways
  21. 21. Glucose Glucose-6-phosphate ATP ADP Heterolactic Fermentation: Pentose-PO 4 entry Ribulose-5-phosphate CO 2 O 2 NAD + NADH + H + NAD + NADH + H + H 2 O 2 2 H 2 O NADH oxidase NADH oxidase 1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate ( PEP ) Pyruvate Lactate ATP ADP H 2 PO 4 - NAD + NADH + H + (2) H 2 O ATP ADP NAD + NADH + H + LDH 6-phospho-gluconate NAD + NADH + H + Xylulose-5-phosphate NAD + NADH + H + Products : 2 ATP 1 Lactate 1 Acetate Glyceraldehyde-3-phosphate H 2 PO 4 - Acetyl-phosphate Phosphoketolase ATP ADP Acetate
  22. 22. L.A.B. groups based on fermentation pathways <ul><li>Obligately homofermentative </li></ul><ul><li>Obligately heterofermentative </li></ul><ul><li>Facultatively heterofermentative </li></ul>
  23. 23. Obligately homofermentative Group I Lactobacillus species and a few other species <ul><li>Hexoses are fermented by homolactic fermentation </li></ul><ul><li>pathway (glycolysis + LDH) </li></ul><ul><li>Can do mixed acid fermentation of hexoses under certain conditions </li></ul><ul><li>Do not ferment pentoses </li></ul><ul><li>Have FDP aldolase enzyme </li></ul><ul><li>Do not have phosphoketolase enzyme </li></ul>
  24. 24. Homolactic Fermentation of Glucose (Glycolysis + LDH) Glucose Fructose-1,6-diphosphate ATP ADP (2) 3-Phosphoglycerate (2) ATP (2) ADP (2) 1,3-Diphosphoglycerate (2) H 2 PO 4 - (2) NAD + (2) NADH + (2) H + (2) 2-Phosphoglycerate (2) Phosphoenolpyruvate ( PEP ) (2) H 2 O (2) Pyruvate (2) ATP (2) ADP (2) Glyceraldehyde-3-phosphate Dihydroxyacetone-phosphate FDP aldolase Products : 2 ATP 2 Lactate Key enzymes : FDP aldolase Lactate dehydrogenase (2) Lactate (2) NAD + (2) NADH + (2) H + Lactate dehydrogenase (LDH) cofactor regeneration O-P-O O O phosphate group Glucose-6-phosphate ATP ADP O-P-O O O Fructose-6-phosphate
  25. 25. Glucose Heterolactic Fermentation of Glucose --- Aerobic conditions O 2 Glucose-6-phosphate ATP ADP 1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate ( PEP ) Pyruvate Lactate ATP ADP H 2 PO 4 - NAD + NADH + H + (2) H 2 O ATP ADP NAD + NADH + H + LDH 6-phospho-gluconate NAD + NADH + H + Xylulose-5-phosphate Ribulose-5-phosphate NAD + NADH + H + CO 2 Products : 2 ATP 1 CO 2 1 Lactate 1 Acetate Key enzymes : Phosphoketolase Lactate dehydrogenase NADH oxidase Glyceraldehyde-3-phosphate H 2 PO 4 - Acetyl-phosphate Phosphoketolase ATP ADP Acetate NAD + NADH + H + NAD + NADH + H + H 2 O 2 2 H 2 O NADH oxidase NADH oxidase
  26. 26. Obligately heterofermentative Group III Lactobacillus species, Leuconostoc , Oenococcus <ul><li>Hexoses are fermented by heterolactic fermentation </li></ul><ul><li>pathway (phosphoketolase pathway) </li></ul><ul><li>Pentoses are fermented by heterolactic fermentation </li></ul><ul><li>pathway (phosphoketolase pathway) </li></ul><ul><li>Have phosphoketolase enzyme </li></ul><ul><li>Do not have FDP aldolase enzyme </li></ul>
  27. 27. Facultatively heterofermentative Group II Lactobacillus species, Lactococcus , Pediococcus , Streptococcus thermophilus <ul><li>Hexoses are fermented by homolactic fermentation </li></ul><ul><li>pathway (glycolysis + LDH) </li></ul><ul><li>Can do mixed acid fermentation of hexoses under certain conditions </li></ul><ul><li>Pentoses are fermented by heterolactic fermentation </li></ul><ul><li>pathway (phosphoketolase pathway) </li></ul><ul><li>Have both FDP aldolase and phosphoketolase </li></ul><ul><li>enzymes </li></ul>
  28. 28. Lb. rhamnosus Lactobacillus Homolactic and facultatively heterolactic – no CO 2 from glucose, FDP aldolase present Obligately heterolactic – CO 2 from glucose, phosphoketolase present Lb. sanfrancisco Lb. kefir
  29. 29. Citrate Transport Citrate H + Citrate H + Citrate Permease Citrate permease is pH dependent – only functions pH 5 – 6. Optimum = pH 5.2 ~1.5 mg/ml citrate in milk CH 2 - COOH CH 2 - COOH HO - C - COOH
  30. 30. Citrate Metabolism Acetate CO 2 Leuc. mesenteroides subsp. cremoris Lc. lactis subsp. lactis biovar. diacetylactis citrate lyase Pathway does not generate ATP, but regenerates NAD + . citrate NAD + NADH + H + NAD + NADH + H + CH 2 - COOH CH 2 - COOH HO - C - COOH CH 3 – C – C – CH 3 O O
  31. 31. Lactate Export Sugar Lactate - Malate - Antiport Symport Lactate - 2H + 2H + (Not common)
  32. 32. Exopolysaccharide Synthesis Some strains of Lc. lactis, S. thermophilus, Leuconostoc, Lactobacillus
  33. 33. Exopolysaccharide types and applications <ul><li>Capsular: yogurts (thickener), cheeses (increase moisture) </li></ul><ul><li>Ropy: some fermented milks such as viili and l å ngfil </li></ul>(Perry et al. 1997. Journal of Dairy Science. 80:799-805.)
  34. 34. L.A.B.: Carbohydrate Metabolism Transport of sugars across cell membrane Catabolism of sugars for energy Synthesis of polysaccharides Homolactic fermentation Mixed acid fermentation Heterolactic fermentation <ul><li>Pathways for glucose fermentation </li></ul><ul><li>Pathways for other hexoses </li></ul><ul><li>Pathways for pentoses </li></ul><ul><li>Pathway for citrate </li></ul><ul><li>Disaccharides </li></ul>

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