Chapter 22 : Carbohydrate Fermentations

TABLE 1

Glucose fermentation by Clostridium butylicum: calculation of carbon recovery, O/R balance, and available hydrogen balance

a Data in this column are taken from reference 123 .

b This value is the product of the number of carbons × the number in column [2]. Carbon recovered = 578/600 × 100 = 96.3%; O/R balance = 408/384 = 1.06; balance of available H = 2,400/2,264 = 1.06.

c This value is the product of column [2] × column [4].

d Calculation of available [H] is based on the following equations: C6H12O6 + 6H2O → 24H + 6H2O; C4H8O2 + 6H2O → 20H + 4CO2; C2H4O2 + 2H2O → 8H+ 2CO2; C4H10O + 7H2O → 24H + 4CO2; C3H8O + 5H2O → 18H + 3CO2.

e This value is the product of column [2] × column [6].

TABLE 2

Assay procedures for enzymes of the EMP pathway a

a Most reagents and enzymes described are readily available from various commercial sources, such as Sigma-Aldrich.

TABLE 3

Assay procedures for the enzymes of the ED pathway

a BSA, bovine serum albumin; MES, morpholineethanesulfonic acid.

TABLE 4

Assay procedures for enzymes of the homolactate fermentation pathway

a Preparation of permeabilized cells: Bacterial culture is washed twice with ice-cold 50 mM KPO4 buffer (pH 6.5) containing 2 mM MgSO4 (KPM buffer), resuspended in 1/100 of culture volume of KPM buffer containing 20% (wt/vol) glycerol, and rapidly frozen in liquid nitrogen and kept at –80°C until use. After thawing,cells are washed once with KPM buffer; resuspended in 50 mM KPO4 buffer (pH 6.5) containing 12.5 mM NaF, 5 mM MgCl2, and 2.5 mM dithiothreitol (DTT); and permeabilized. First, 2.5 μl of toluene/acetone (1:9, vol/vol) is added per 250 μl of cell suspension and vortexed for 5 min at 4°C. The cells are then centrifuged (150×g at 4°C for 2 min) and the cell pellet is resuspended in the same buffer (optical density at 600 nm of 50) and again treated with toluene/acetone as described above ( 50 ). After 5 min of vortexing, permeabilized cells are kept on ice.

b Separation method: The product, [14C]hexose-phosphate, is separated from excess 14C-substrate by ion-exchange chromatography. The incubation mixtures are diluted with 0.5 ml of water and transferred to columns, 0.8 by 9 cm, of analytical-grade, chloride form, ion-exchange resin (AG l-X2, 50 to 100 mesh; Bio-Rad); the excess 14C-substrate is washed from each column with 15 ml of water; and the labeled product is eluted from the column with 6 ml of 1.0 M LiCl. Each elute is collected in a liquid scintillation spectrometer vial, and the 14C is counted after the addition of 15 ml of a mixture containing 333 ml of Triton X-100 (Packard Instrument Company), 666 ml of toluene, 5.5 g of 2,5-diphenyloxazole, and 125 mg of dimethyl-1,4-bis[2-(5-phenyloxazolyl)] benzene (dimethyl POPOP). The efficiency of the counting system is determined with 14C-standards (see chapter 17).

TABLE 5

Assay procedures for enzymes of the heterolactate fermentation pathway

a CHES, 2(N-cyclohexylamino) ethanesulfonate; DTT, dithiothreitol.

TABLE 6

Assay procedures for enzymes of the heterofermentative bifidum pathway

TABLE 7

Assay procedures for the enzymes of the butyrate-butanol-acetone-isopropanol fermentation in C. acetobutylicum

a DTT, dithiothreitol; KF, potassium fluoride; GSH, glutathione (reduced); FAD, flavin adenine dinucleotide; MOPS, morpholinepropanesulfonic acid.

TABLE 8

Assay procedures for the enzymes involved in ethanol and acetate fermentation by C. kluyveri

a FAD, flavin adenine dinucleotide; RS, regenerating system.

b H2 is quantified by gas chromatography. Sample column: Length, 4 m; inner diameter, 2 mm; material, steel; temperatures, 50°C for both injection port and column;carrier gas, argon; detection, thermal conductivity detector; gas samples, 2 ml of 15-ml gas phase is injected with a gastight syringe.

TABLE 9a

Assay procedures for the enzymes of the mixed acid and butanediol fermentation a

a DTT, dithiothreitol; MOPS, morpholinepropanesulfonic acid; BSA, bovine serum albumin.

b The sugar phosphates used are glucose-6-phosphate with [14C]glucose, fructose-1-phosphate with [14C]fructose, and mannitol-1-phosphate with [14C]mannitol. The resin used for separating 14C-sugar from 14C-sugar-phosphate is AG1-X2, 50- to 100-mesh size, chloride form.

c The tube is evacuated and filled with O2-free argon, and 10 μl of 20 mM Fe(NH4)2(SO4)2 is then added to the first arm. The tube is preincubated under argon for 30 min. The contents of the two arms are mixed and the tube is illuminated by a daylight lamp. The enzyme is fully activated usually within 30 min. The larger chloroplast fragments are centrifuged and the mixture is stored at 0°C. The low-molecular-weight components of the activated enzyme solution are removed by gel filtration under argon with a Sephadex G-25 column (1.6 by 20 cm) with an anaerobic buffer containing 50 mM MOPS (pH 7.6), 9 mM DTT, and 0.2 mM Fe(NH4)2(SO4)2. Temperature, 4°C; flow rate, 2 ml/min. The protein fraction is collected into argon-flushed tubes and stored at 0°C.

TABLE 9b

Assay procedures for the enzymes of the mixed acid and butanediol fermentation a

a DTT, dithiothreitol; MOPS, morpholinepropanesulfonic acid; BSA, bovine serum albumin.

b The sugar phosphates used are glucose-6-phosphate with [14C]glucose, fructose-1-phosphate with [14C]fructose, and mannitol-1-phosphate with [14C]mannitol. The resin used for separating 14C-sugar from 14C-sugar-phosphate is AG1-X2, 50- to 100-mesh size, chloride form.

c The tube is evacuated and filled with O2-free argon, and 10 μl of 20 mM Fe(NH4)2(SO4)2 is then added to the first arm. The tube is preincubated under argon for 30 min. The contents of the two arms are mixed and the tube is illuminated by a daylight lamp. The enzyme is fully activated usually within 30 min. The larger chloroplast fragments are centrifuged and the mixture is stored at 0°C. The low-molecular-weight components of the activated enzyme solution are removed by gel filtration under argon with a Sephadex G-25 column (1.6 by 20 cm) with an anaerobic buffer containing 50 mM MOPS (pH 7.6), 9 mM DTT, and 0.2 mM Fe(NH4)2(SO4)2. Temperature, 4°C; flow rate, 2 ml/min. The protein fraction is collected into argon-flushed tubes and stored at 0°C.

TABLE 10

Assay procedures for the enzymes of the acrylate pathway for propionate production

a BSA, bovine serum albumin.

TABLE 11

Assay procedures for the enzymes of the succinate pathway for propionate production

a DBC [(5,6-dimethyl benzimidazolyl) Co-5′-deoxyadenosine cobamide] should be stored in a light-proof container. Benzimidazolylcobamide or adenylcobamide can also be used as coenzymes.

TABLE 12

Assay procedures for the enzymes of the bacterial acetate fermentation pathway

a DTT, dithiothreitol.

b All reagents are prepared anaerobically by boiling the water containing reagents and storing them under nitrogen. The assay is performed in nitrogen-filled, serum-stoppered anaerobic cuvettes and the enzyme is added via a syringe. NAD+ reduction is monitored by measuring the absorbance at 340 nm, and methyl viologen reduction is measured at 600 nm.

c The reaction is initiated with the addition of acetyl-CoA and is allowed to proceed for 15 min at 55°C. The reaction is stopped by adding 0.04 ml of 4 M acetic acid, which drops the pH to 3.5. The solution is then made alkaline with the addition of 0.2 ml of 2 N NaOH to hydrolyze acetyl-CoA over a period of 6 h at room temperature. Carrier acetic acid (0.0032 mM) is added and the acetate is then isolated using chromatography on Celite before assaying for 14C.

TABLE 13

Phosphoryl donors for kinases of archaea a

a ADP-GK, ADP-glucokinase; ATP-HK, ATP-hexokinase.

b In M. jannaschii, ADP-GK and ADP-PFK activities are carried out by a single bifunctional enzyme.

TABLE 14

Assay procedures for the enzymes of the acetate fermentation pathway in archaea

a DHAP, dihydroxyacetone phosphate; EPPS, N-(2-hydroxyethyl) piperazine-N9-3-propanesulfonic acid; ACS, acetyl-CoA synthetase.