Tema 10A: Fermentations. Chapter 14 and Chapter 8 Pages

Transcription

1 Tema 10A: Fermentations hapter 14 and hapter 8 Pages

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3 Lactic Acid Bacteria haracteristics: Gram positive, carbohydrate users, proteolysis rare, nonmotile, non-spore forming Strict fermentors, unable to synthesize cytochromes unless heme is added. catalase negative oxidase negative Nutritionally fastidious All make lactic acid (lactate) as predominant end product

4 Lactic Acid Bacteria Types of fermentation omofermentative: glucose to 2 lactic acids, 85-95% of glucose carbon in lactate eterofermentative: glucose to 1 lactate, 1 ethanol, and 1 carbon dioxide, only 50% or less of glucose carbon in lactate. Types of products will define the pathway used and ATP made.

5 Lactic Acid Bacteria Types of organisms Streptococcus: homofermentative Leuconostoc: heterofermentative Pediococcus homofermentative Lactobacillus; heterofermentative or homofermentative.

6 Lactic Acid Bacteria Streptococcus species: Group A, flesh eating, toxin (superantigens and pyrogenic) Pneumonococci: polysaccharides Enterococcus: gut dwellers Lactococcus natural fermentations Lactic acid production: lowers p, preserves and precipitates proteins

7 Lactic acid bacteria omofermentative pathway Uses Glycolytic pathway to make 2 pyruvates from glucose verview: Activation-use 2 ATP Make ß-carbonyl - bond cleavage xidation/reduction Substrate-level phosphorylation

8 omofermentative Pathway in Streptococci Glucose ATP ADP Glucose-6-P Fructose-6-P ATP ADP Fructose-1,6 bis P Lactate dehydrogenase Dihydroxyacetone-P Dihydroxyacetone-P converted to glyceraldehyde-3-p Pathway shows 2 G-3-P's after this step. Reoxidation of NAD 2 pyruvate 2 NAD 2 NAD + 2 lactate Used 2 ATP Made 4 ATP Net ATP yield=2 ATP/glucose 2 Glyceraldehyde-3-P 2 NAD + 2 NAD 2 1,3-bisphosphoglycerate 2 ADP 2 ATP 2 3-phophoglycerate 2 2-phosphoglycerate phosphoenolpyruvate 2 ADP 2 ATP 2 pyruvate

9 Glycolytic Pathway for Glucose Metabolism 2 exokinase or PTS system = 3 P ATP 2 G6P isomerase ADP Glucose Glucose-6-P = 3 P Phosphofructokinase = 3 P Fructose-1,6-bis P aldolase = 3 P 2 2 Glyceraldehyde-3-P 2 + ATP ADP 2 P 3 = 2 Dihydroxyacetone-P Fructose-6-P Fructose-1,6- bisp 2 P = 3 Now have 2 G3P's Triose isomerase

10 Triosephosphate dehydrogenase Glycolytic Pathway for Glucose Metabolism = 3 P 2 NAD + NAD { = 3 P 2 S-Enz } P = 4 Glyceraldehyde-3-P (Metabolism of only one G3P is shown) Phosphoglycerate kinase = 3 P 2 -P = 3 ADP ATP = 3 P 2 1,3-bisphosphoglycerate 3-phosphoglycerate Phosphoglycerate mutase Enolase P 3 = -P 3 = 2-phosphoglycerate phosphoenolpyruvate Pyruvate kinase ADP ATP 3 From the 1 and 6 carbons of glucose pyruvate From the 3 and 4 carbons of glucose

11 Isomerization Reaction: reates an electron attracting keto group at the # 2 carbon Mechanism of the isomeration reaction 2 P3 = Glucose-6-P 2 P3 = cis-enolate 2 P3 = Fructose-6-P dissociates from 2 2 electrons shift to form cis enediol from hydroxyl group dissociates 2 electrons shift to form keto group. Forces electrons in enol bond to shift to 1.

12 - bond cleavage: Aldolase Reaction Mechanism of the aldolase reaction 2 P 3 = Enol formation 2 P 3 = P 3 = Dihydroxyacetone-P 2 P 3 = arbonyl beta to arbon with 2 P 3 = Glyceraldehyde-3-P dissociates from 4; 2 electrons shift to form cis enediol from hydroxyl group (4) dissociates 2 electrons shift to form keto group. Forces electrons in enol bond to shift to 1.

13 In conclusion Streptococcus Uses glycolysis to degrade glucose to 2 pyruvates NAD s made in pathway are reoxidized by reducing pyruvate to lactate NAD is key cofactor in oxidation reduction reactions ATP made solely by substrate level phosphorylation.

14 eterofermentative Pathway Uses part of the pentose phosphate pathway nly one pyruvate is made ave a decarboxylation and - cleavage to give a 3 and a 2 verview: Activation-use 1 ATP Two oxidations done to make ß-carbonyl - bond cleavage G3P to pyruvate like in Streptococcus Less ATP because more ox/red reactions

15 eterofermentative Pathway in Leuconostoc sp. Glyceraldehyde-3-P PGALD dehydrogenase NAD+ NAD 1,3-bisphosphoglycerate ADP PGA kinase ATP 3-phophoglycerate PGA mutase Glucose ATP hexokinase ADP Glucose-6-P NADP+ G6P dehydrogenase NADP [6-P-Gluconolactone] 2 6-P-gluconate NADP+ 6PG dehydrogenase 2 NADP Ribulose-5-P R5P epimerase Xylulose-5-P 2-phosphoglycerate enolase 2 phosphoenolpyruvate ADP pyruvate kinase ATP pyruvate lactate dehydrogenase phosphoketolase NAD NAD + Lactate Ethanol NADP+ NADP alcohol dehydrogenase Acetaldehyde NADP + acetaldehide dehydrogenase NADP Acetyl-oA P i phosphotransacetylase oa Acetyl-P ADP Acetate ATP What would happen if the organism could Small divert electrons away from ethanol production? amount ATP yield used 1 ATP made 2 ATP net yield= 1 ATP Additional oxidation/reduction reactions decrease potential ATP yield.

16 2 P = 3 Glucose-6-P NADP + NADP G6P dehydrogenase 2 P = 3 6-P-glucono-lactone (enzyme-bound) 6-P-gluconate 2 2 P = 3 NADP + 6PG dehydrogenase NADP P = 3 Ribulose-5-P 6PG dehydrogenase 2 P = 3 3-keto-6-P-gluconate (enzyme-bound)

17 Mechanism of beta-decarboxylation ) arbonyl accepts electrons from - between the 1 and 2 carbons. 2) arbon dioxide and an enolate are formed. 3) Re-shifting of the electrons forms the keto sugar.

18 Phosphoketolase Reaction 2 2 P 3 = -P 3 = 3 Acetyl-Phosphate -P = 3 + Glyceraldehyde-3-P 2 P = 3 Xylulose-5-P Enzyme contains Thiamin pyrophosphate (TPP) as cofactor the function here is transketolation. E-TPP- 2 E-TPP 2 -P 3 = Acetyl-Phosphate

19 onclusion eterofermentative organisms use a pathway with a greater number of redox reactions than Streptococcus. Make very oxidized and very reduced compounds. More NAD(P) to be reoxidized constrains ATP synthesis, high energy intermediate used as an electron acceptor. Vitamins: essential portions of cofactors that organism can not make de novo

20 Fermentation Analysis In order to understand how an organism makes its energy or what biochemical pathways are present, one must first know what the products of metabolism are. First Law of Thermodynamics: mass is conserved must account for all of the carbon and electrons originally present in the substrate.

21 Fermentation analysis From this, we can then figure out the pathways and amount of ATP made. Also, inspection of the products will allow us to make predictions about the cell s metabolism. Initially, we will look at glucose consumption in rich medium Growth factors from media supply cell carbon Most of glucose goes to products, only 5-10% incorporated into cells. In industry, one must also account for cell mass.

22 Experimental set up Glucose added and inoculated ontrol: inoculated but without glucose; correct for products made from other medium components or brought in with inoculum. Take time zero and time final samples and measure Glucose and product formation.

23 Example: Leuconostoc brevis ompd. Amount (mmol) # of s mmol of Glucose Lactate Glycerol Ethanol Acetate

24 ave we detected all of the products? carbon alculate the carbon recovery by multiplying the amount detected by the number of carbon atoms for each compound, then sum up all of the carbon in the products. arbon in glucose = 6 X 100 mmoles =600 mmoles arbon in products = ( ) mmoles arbon in products = mmoles % recovery = (584.7 mmol/600 mmol) * 100 % recovery = 97.4%

25 ave detected all of the electrons? In a fermentation, electrons removed from glucose are added back to a compound derived from glucose. Thus, the ratio of oxidized products to reduced products must equal 1. Since glucose ( ) has 2 s for every, products with more than 2 s per have been reduced, and products with less than 2 s per have been oxidized.

26 R value of a compound To calculate the R value of a compound, give a numerical score of +1 for every and -1 for every 2 s. Examples: Glucose ( ): 6 is +6, 12 's is -6, 6-6=0 Lactate ( ): 3 is +3, 6's is -3, 3-3=0 Acetate ( ): 2 is +2, 4's is -2, 2-2=0 Glycerol ( ): 3 is +3, 8 's is -4, 3-4 = -1 Ethanol ( 2 6 ): 1 is +1, 6 is -3, 1-3= -2 arbon dioxide ( 2 ): 2 's = +2

27 Example: Leuconostoc brevis ompd Amount (mmol) R value mmol (ox) mmol (red) Glucose Lactate Glycerol Ethanol Acetate

28 /R ratio of the fermentation nce the R value of the compound is determined this is multiplied by the amount detected (see Table) alculate the /R ratio R ratio = 178.6/(-171.8)+(-6.8) R ratio = 178.6/178.6 = 1.0 Ratios close to 1 mean all of the electrons have been accounted for.

29 1 to 2 ratio A common - cleavage reaction is 3 --> usually indicating pyruvate is an intermediate. If this occurs in your organism, then expect a 1 / 2 ratio of 1. 1 = 89.3 mmoles 2 = 85.9 mmoles mmoles = 93.2 mmoles 1 to 2 ratio = 89.3 mmoles/ 93.2 mmoles 1 to 2 ratio = 0.96 Value is close to one so probably have pyruvate cleavage.

30 onclusion Fermentation balance is the first step in understanding the metabolism of an organism Must have recovery close to 100% and an /R ratio close to 1. 1 / 2 ratio indicates pyruvate cleavage You can use the above information in the lab to determine what analyses are needed to complete the balance.

31 What happens if an alternate electron acceptor is present in a fermentation? Electron flow dictates carbon flow and energy yield Alternate electron acceptors provide fermentative bacteria a choice The result will be less lactate and ethanol and more acetate and ATP are made. We will study the effect of oxygen on the metabolism of lactic acid bacteria De Felipe et al., J. Bacteriol. vol 180, p 3804, 1998

32 Utilization of oxygen by facultative lactic acid bacteria. Some lactic acid bacteria possess enzymes that reoxidize NAD (and NADP) by reducing oxygen to water (Dolin s enzymes) xidase NAD(P) > NAD(P) + Peroxidase NAD(P) > NAD(P) +

33 What happens when oxygen is present? When oxygen and Dolin s enzymes are present, NAD(P) is reoxidized by reducing oxygen to water rather than pyruvate to lactate or acetyl-p to ethanol. More acetate and ATP, less ethanol and lactate, are made.

34 Make more ATP Acetate kinase acetyl-p + ADP --> acetate + ATP For every acetate made, one ATP is made by substratelevel phosphorylation by this reaction. When Dolin s enzymes and oxygen are present, 1) acetyl-p goes to acetate and ATP rather than to ethanol, and 2) pyruvate is metabolized to acetate and 2 rather than to lactate. 2 Pyruvate Acetyl-oA Acetyl-P Acetate NAD + NAD oa P i ADP ATP

35 Streptococcus sp. and Dolin s enzymes No 2 or Dolin's enzymes Glucose 2 NAD + With 2 and Dolin's enzymes Glucose 2 NAD + 2 ATP net 2 NAD 2 ATP net 2 NAD 2 Pyruvate 2 NAD 2 Pyruvate 2 oa 2 NAD + 2 NAD + 2 Lactate 2 2 NAD 2 2 Acetyl-oA NAD 2 P i 2 oa 2 Acetyl-P 2 ADP 2 ATP NAD + Net of 4 ATP 2 Acetate

36 Summary If there is an alternate electron acceptor, less lactate, more acetate, 2, and ATP

37 Bifidobacterium sp. Bifid or 2 lobes Gram positive, curved rod found in the feces of breast-fed infants, Requires many growth factors, including N-acetylglucosamine Makes 2 lactate and 3 acetate from 2 glucose Makes high-energy intermediate by phosphoketolase reaction rather than ox/red. Matsuki, Takahiro, Koichi Watanabe, Ryuichiro Tanaka, and iroshi yaizu, 1998, "Rapid Identification of uman Intestinal Bifidobacteria by 16S rrna-targeted Species- and Group-Specific Primers," FEMS Microbiology Letters, Vol. 617, pp Bonjoch, X., E. Ballesté, and A. R. Blanch. (2004) Multiplex PR with 16S rrna Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution. AEM V70(5): Schell, M. A. et. al. (2002). The genome sequence of Bifidobecterium longum reflects its adaptation to the human gastrointestinal tract. PNAS V99 N22 p

38 utline of pathway Activation: 2 glucose to 2 fructose-6-p uses 2 ATP Make 2 G3P and 3 acetyl-p from 2 glucose by transketolase, transaldolase, and phsophoketolase reactions 2 G3P to 2 lactate by reactions seen in Streptococcus 3 acetyl-p to 3 acetate and 3 ATP by acetate kinase

39 Synthesis of 2 G-3-P and 3 2 units from 2 glucose. Phosphoketolase : 6 -> 4 + acetyl-p Transaldolase: > Transketolase: > Phosphoketolase: > acetyl-p Net Result: 2 6 -> acetyl-p Uses these enzymes to interconvert hexoses and pentoses.

40 3 -P 3 P i Phosphoketolase: 6 (or 5 ) + P i -> 4 (or 3 ) + acetyl-p + + Transaldolase: > Transketolase > 5 + 5

41 2 glucose -> 2 lactate + 3 acetate 2 Glucose 2 ATP 2 ADP Fructose-6-P + Fructose-6-P Phosphoketolase P i Acetyl-P Erythrose-4-P Transaldolase Glyceraldehyde-3-P Sedoheptulose -7-P 2 lactate 2 NAD + 2 NAD 2 pyruvate 2 ATP 2 ADP 2 phosphoenolpyruvate phosphoglycerate 2 3-phosphoglycerate 2 ATP Xylulose-5-P Ribose-5-P 2 Acetyl-P 2 ADP 2 1,3-bisphosphoglycerate Ribulose-5-P 2 NAD 2 Xylulose-5-P 2 NAD + 2 Glyceraldehyde-3-P hapter 8, Fig Acetyl-P 3 ADP 3 ATP 3 Acetate ATP yield: (7-2)/2 glucose = 2.5 ATP/ glucose

42 Summary Make acetyl-p by phosphoketolase rather than by ox/red reaction Don t have to use acetyl-p as electron acceptor ATP yield higher than other anaerobes for this reason. Avoidance of ox/red leads to higher ATP gain.