4.2 Methane Metabolism in Methane-Oxidizing Bacteria
87
4.2.3.2 MDH Inhibition
The dehydrogenation of methanol to formaldehyde is catalyzed by PQQ-dependent
MDH. The electrons generated in this reaction are donated to the terminal oxidase of
methane-oxidizing bacteria via cytochrome c L [30, 78, 79]. Since this dehydrogenation reaction is faster than the oxidation reaction of methane to methanol, only trace
concentrations of methanol are observed in the reaction mixture unless methanol
dehydrogenation is suppressed [17, 87, 115].
The most widely used strategies for the inhibition of MDH rely on the use of
(i) methane-oxidizing bacteria whose MDH is inhibited in advance by chemical
modification followed by its use for methane oxidation reaction and (ii) methaneoxidizing bacteria for methane oxidation reactions in the presence of MDH inhibitors.
Cyclopropanol [10, 11] is used in the former strategy, and phosphate [7, 8, 16, 19,
22, 24–26], NaCl [12, 15, 17–19], EDTA [15, 19], MgCl 2 [7, 8, 16, 17, 19, 22, 24,
25], and NH 4 Cl [17, 19] are used in the latter. These inhibitors can be used alone or
in combination.
(1) Cyclopropanol
Cyclopropanol combines with a coenzyme of MDH, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinolone-4,5-dione (PQQ), resulting in irreversible deactivation. Cyclopropanol has been reported to be the most efficient MDH inhibitor. It is considered
an irreversible inhibitor because it produces stable compounds from interaction with
free and MDH-bound PQQ [15, 17, 109]. The optimum pyrrole concentration is
0.67 μM; MDH activity is reduced by 50% at this concentration, whereas MMO
activity decreases by only 5.2% [10, 11, 15, 17, 116]. However, cyclopropanol is
not used universally because of its difficult preparation and instability under aerobic
conditions.
(2) Salt
Salts such as NaCl, NH 4 Cl, and phosphate disrupt electron transport, which
in turn decreases the activity of MDH [115] by weakening the ionic interaction
between MDH and cytochromes c L . Phosphate is the most widely used inhibitor of
methanol oxidation, and is typically used in the concentration range 40–100 mM [7,
8]. This form of inhibition is considered non-competitive and reversible [7, 8]. NaCl
is advantageous as an inhibitor for methanol production because of its stability, abundance, and low cost. According to some studies, the optimum NaCl concentration
for methanol production is approximately 200 mM [12], although NaCl concentrations exceeding 100 mM distort the cell morphology. NH 4 Cl is an effective MDH
inhibitor, and high conversion efficiency from methane to methanol is observed when
40 mM NH 4 Cl is used. However, the methanol yield is low [15, 19, 22, 24], because
NH 4 Cl also inhibits the oxidation of methane to methanol by MMO [15, 19, 22, 24].
Therefore, NH 4 Cl is not a suitable MDH inhibitor for the methanol synthesis reaction.
87
4.2.3.2 MDH Inhibition
The dehydrogenation of methanol to formaldehyde is catalyzed by PQQ-dependent
MDH. The electrons generated in this reaction are donated to the terminal oxidase of
methane-oxidizing bacteria via cytochrome c L [30, 78, 79]. Since this dehydrogenation reaction is faster than the oxidation reaction of methane to methanol, only trace
concentrations of methanol are observed in the reaction mixture unless methanol
dehydrogenation is suppressed [17, 87, 115].
The most widely used strategies for the inhibition of MDH rely on the use of
(i) methane-oxidizing bacteria whose MDH is inhibited in advance by chemical
modification followed by its use for methane oxidation reaction and (ii) methaneoxidizing bacteria for methane oxidation reactions in the presence of MDH inhibitors.
Cyclopropanol [10, 11] is used in the former strategy, and phosphate [7, 8, 16, 19,
22, 24–26], NaCl [12, 15, 17–19], EDTA [15, 19], MgCl 2 [7, 8, 16, 17, 19, 22, 24,
25], and NH 4 Cl [17, 19] are used in the latter. These inhibitors can be used alone or
in combination.
(1) Cyclopropanol
Cyclopropanol combines with a coenzyme of MDH, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinolone-4,5-dione (PQQ), resulting in irreversible deactivation. Cyclopropanol has been reported to be the most efficient MDH inhibitor. It is considered
an irreversible inhibitor because it produces stable compounds from interaction with
free and MDH-bound PQQ [15, 17, 109]. The optimum pyrrole concentration is
0.67 μM; MDH activity is reduced by 50% at this concentration, whereas MMO
activity decreases by only 5.2% [10, 11, 15, 17, 116]. However, cyclopropanol is
not used universally because of its difficult preparation and instability under aerobic
conditions.
(2) Salt
Salts such as NaCl, NH 4 Cl, and phosphate disrupt electron transport, which
in turn decreases the activity of MDH [115] by weakening the ionic interaction
between MDH and cytochromes c L . Phosphate is the most widely used inhibitor of
methanol oxidation, and is typically used in the concentration range 40–100 mM [7,
8]. This form of inhibition is considered non-competitive and reversible [7, 8]. NaCl
is advantageous as an inhibitor for methanol production because of its stability, abundance, and low cost. According to some studies, the optimum NaCl concentration
for methanol production is approximately 200 mM [12], although NaCl concentrations exceeding 100 mM distort the cell morphology. NH 4 Cl is an effective MDH
inhibitor, and high conversion efficiency from methane to methanol is observed when
40 mM NH 4 Cl is used. However, the methanol yield is low [15, 19, 22, 24], because
NH 4 Cl also inhibits the oxidation of methane to methanol by MMO [15, 19, 22, 24].
Therefore, NH 4 Cl is not a suitable MDH inhibitor for the methanol synthesis reaction.