Anthraquinone-2,6-disulphonate

Anthraquinone-2,6-disulphonic acid (disodium salt) [84-50-4] M 412.3, m >325°. Crystd three times from water, in the dark [Moore et al. JCSFTl 82 745 1986]. 

Disodium anthraquinone-2,6-disulphonate [853-693-9] M 412.3. Crystd from water. 

For dehydrogenations very different electron mediators can be used. For example anthraquinone-2,6-disulphonate is very effective. 

Besides the artificial mediators mentioned so far safranine T in 0.75 mM concentration shows about 80 % of the activity observed with 1 mM methylviologen or 1 mM anthraquinone-2,6-disulphonate for the reduction of 2-methylfumarate. The absence of a mediator decreases the reduction rate to 3-4 %. C. formicoaceticum is also useful for stereoselective hydrations of fumarate and maleate derivatives (36). 

Scheme 4 Reductions of 2-oxo carboxylates to (R)-2-hydroxy carboxylates and dehydrogenations of (R)-2-hydroxy carboxylates to 2-oxo carboxylates. Properly grown Proteus cells contain hydrogenase and formate dehydrogenase which can be used to deliver electrons carried by viologens or anthraquinone-2,6-disulphonate to HVOR. The latter carrier also transfers electrons from HVOR to DMSO reductase during dehydrogenations of (R)-2-hydroxy carboxylates.

If the reduction is conducted with benzylviologen (E = -330 to -360 mV (6)) as the mediator at pH 6 the 2-oxo carboxylates will be completely reduced since the equilibrium constant for the reduction of pyruvate as an example is 8.10. At pH 8.5 with anthraquinone-2,6-disulphonate (E" = -184 mV) the equilibriiun constant of this reaction is about 6 orders of magnitude smaller than with benzylviologen and dehydrogenations of (R)-2-hydroxy carboxylates can be conducted quantitatively when the reduced quinone (AQ-2,5-DS-H2) is effectively reoxidized for instance by Reaction [22] ... 

The use of anthraquinone-2,6-disulphonate as mediator is easily possible since P. mirabilis and P. vulgaris possess a DMSO reductase (Scheme 4). Its activity depends on the growth conditions (Table 18), (47,62). 

Another problem was to find a stable and cheap mediator and an effective regeneration system for the oxidized mediator. Table 14 shows the activity of different mediators. Under various aspects anthraquinone-2,6-disulphonate was especially useful. It is completely stable under the reaction conditions and can easily be reisolated if necessary. It works not only veiy well with HVOR but also with dimethyl-sulphoxide reductase present in Proteus mirabilis or P. vulgaris (Scheme 4). 

Space time yield based on 1 g F. vulgaris cells (dry weight). In 0.1 or 0.3 M Tris/HCl-buffer, pH 8.5. Due to the solubility of AQDS higher concentrations are not suitable. Carbamoylmethylviologen. Anthraquinone-2,6-disulphonate. Dimethyl-sulphoxide. iV-Methylmorpholine N-oxide. 

A volume of 50 ml contained 26.0 mmol (/ )-lactate, 0.15 mmol anthraquinone-2,6-disulphonate, 0.25 nunol EDTA, 26.0 mmol DMSO and 3.7 g wet packed cells of P. vulgaris at 40 °C. The pH 8.5 in the slowly stirred suspension was kept constant with 2 M sodiiun hydroxide. The dimethyl-sulphide formed from DMSO (Scheme 4 Reaction [22]) was removed actually completely by a slow stream of nitrogen gas. The boiling point of the dimethyl-sulphide is 38 °C, and its solubility in water is rather low. The formed dimethyl-sulphide was condensed in a cold trap. 

Formation of pyruvate with stoichiometric amounts of anthraquinone-2,6-disulphon-ate as electron acceptor... 

The apparent Km values were determined with crude extracts of P. mirabilis at 38 °C and pH 8.5. The data were calculated according to Eadie and Hofstee (69). The substrates were tested in the presence of 6 mM anthraquinone-2.6-disulphonate (AQDS).Yield as isolated product. Kinetic data for the dimethyl-sulphoxide reductase were determined with 0.6 mM reduced anthraquinone-2.6-disulphonate. The Km values for the mediators were determined with 50 mM D-araWno-hex-2-ulosonate as substrate. mmol kg. (biocatalyst) h . The Km and Vm,x values of the 3 artificial mediators were determined with 50 mM D-ara6/ o-hex-2-ulosonate. 

In crude cell extracts of C. thermoaceticum the specific activity of AMAPORs for NADP reduction with reduced MV is extraordinarily high and more than twice of that for NAD. NADH and NADPH dehydrogenation for the regeneration of NAD and NADP is achieved with the same enzyme activities and anthraquinone-2,6-disulphonate (Reaction [31a]). The activities are about the same as for carbamoylmethylviologen (CAV) (Table 24). 

NAD and NADP regeneration with AMAPOR from Clostridium thermoaceticum and anthraquinone-2,6-disulphonate using oxygen as final electron acceptor... 

In contrast to viologens reduced anthraquinone-2,6-disulphonate formed by Reaction [31a] can be reoxidized in a non-enzymicly catalysed reaction with oxygen as final electron acceptor (Reaction [31b]). The formed hydrogen peroxide can be split by the extremely cheap catalase (Reaction [31c]). The stun of Reactions [31a]-[31c] leads to Reaction [31]. 

This NADP regeneration consists of the three consecutive Reactions [31a]-[31c]. The reduction of anthraquinone-2,6-disulphonate at the expense of NAD(P)H catalysed from AMAPORs in form of crude cell extract of C. thermoaceticum, the reoxidation of the reduced mediator in a spontaneous reaction with oxygen (112) and the splitting of the hydrogen peroxide by catalase. 

Without catalase the yield of 6-phosphogluconate was only 63 % after 5 h and almost the same after 71 h (not shown for 71 h). Hydrogen peroxide formed during the regeneration of anthraquinone-2,6-disulphonate with oxygen (Reaction [31b]) seems to be the reason. The presence of catalase considerably increased the yield. The addition of superoxide dismutase together with catalase did not improve the results. With higher substrate concentrations NADP cycle numbers of above 800 were obtained. 

For the enantioselective dehydrogenation of the ( -enantiomer from a racemic mixture of 3-hydroxybutyrate three different methods were used to regenerate NADP (Table 27) (83). The first method worked with catalytic concentrations of NADP and anthraquinone-2.6-disulphonate. NADPH was reoxidized by oxidized anthraquinone-2.6-disulphonate catalysed by AMAPOR (Reaction [31a]). The reduced anthraquinone-2.6-disulphonate was electrochemically reoxidized. In experiment 2 a method described by the Whitesides group (106) was applied for NADP regeneration. Oxidized anthra-quinone-2.6-disulphonate could also be used in stoichiometric concentrations (exp. 3). In such a case its electrochemical reoxidation was not necessary. Obviously in experiment 1 the product isolation is especially simple. 

Using the purified (5)-3-hydroxybutyrate oxidoreductase from C. tyrobutyricum (107) together with an electromicrobial NADP regeneration system with AMAPOR of C. thermoaceticum crude extracts and anthraquinone-2.6-disulphonate, the (5)-enantiomer of 1.6 mmol (R,5)-3-hydroxybutyrate was dehydrogenated (exp. 1, Table 27). The remaining (R)-3-hydroxybutyrate showed 98 % ee. Application of the NADP -regenera-... 

From Eq. (1.42), it can be inferred that the substrate breaks down into carbon dioxide and proton along with electricity as a by-product. The concept of MFC was demonstrated by Potter in 1910, who used platinum electrodes as well as living cultures of Escherichia coli and Saccharomyces. The anodophile species of the microbes can transfer the electrons directly to the anode. Otherwise, the electron mediators are required in the cell for enhanced power output and increased efficiency. The direct electron transfer to the anode is hindered by a majority of microbial species due to the presence of non-conductive Upopolysaccharides, peptididoglycans and lipid membrane in their outer layers. Hence, mediators are used which capture electrons from the membrane and are reduced. Furthermore, these mediators will again become oxidised once they move across the membrane and release the electrons to the anode. Hence, the electron transfer process keeps the anode replenished which maintains the sustainability of cell. Anthraquinone-2, 6-disulphonate, 2-methylene blue, thionine, 2-hydroxy-l, 4-naphthoquinone, Fe (III) EDTA, Meldola s blue and neutral red are some of the common chemical mediators which enhance electricity production. MFC is known as the... 

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