Amperometric detection of methanol and formaldehyde with biosensor based on methylobacteria biocatalysts.

Kuznetsova T.A.

Tula state university, 300600, Tula, е-mail: tatulyakuz@mail.ru

Aerobic methylobacteria is a special physiological group of microorganisms using methanol and it oxidized and substituted derivatives as an only carbon and energy source. Methylobacteria cells contain a high concentration of methanol- formaldehyde- and formate-dehydrogenases, connected with bacterial respiratory chain and are able to oxidize toxic one-carbon compounds. Membrane localization of dehydrogenases makes the interaction of methylobacteria cells with artificial electron acceptors easier that enabled to use it as biocatalysts in mediator amperometric biosensors. Such systems can be used during biotechnological processes based on methylotrophs for monitoring initial substrate and intermediates, for determining the concentration of C₁ compounds in waste waters.

The work objective is development of amperometric mediator biosensor based on methylobacteria biocatalysts for the selective detection of methanol and formaldehyde.

In this work we used aerobic methylobacteria Methylobacterium genus, obtained from Doronina N.V. (Institute of Biochemistry and Physiology of Microorganisms, Pushchino). Electrochemical measurements were carried out using a potentiostat-galvanostat IPC-Micro 8.65 «Kronas» (Russia), integrated with the PC. The work mediator electrode was filled with graphite paste, which contained 10% ferrocene and immobilized biocatalysts (cells, enzyme fraction and MDH of methylobacteria) on the surface.

Taken into account the cytoplasmic (formaldehyde dehydrogenase) and membrane (methanol dehydrogenase (MDH)) localization of dehydrogenases we decided to use enzyme fraction of methylobacteria as biocatalysts in biosensor. Cytoplasmic and membrane fractions was obtained by differential centrifugation of disintegrated cells using Avanti J-30I «Beckman Coulter, Inc.» (USA). Sensors based on cytoplasmic and membrane fractions were characterized by a substantial increase of the relative responses for formaldehyde (FA) in comparison with cells sensor (Fig. 1).

Fig. 1. Substrate specificity of cells and enzyme fractions of

M. dichloromethanicum DM4 obtained by ameprometric mediator biosensor with ferrocene as an electron acceptor.

Since the selectivity of biosensor assay is determined by the specificity of biological material, the using of membrane fraction M. dichloromethanicum enabled to carry out selective analysis of the FA level in the presence of methanol and other alcohols.

MDH M. nodulans showed a wide substrate specificity range regarding primary alcohols, but it did not oxidize secondary and benzyl alcohols (Fig. 2).

Fig. 2. Substrate specificity of MDH M. nodulans ORS2060T under homogeneous conditions in the presence of FMS and DCPIP.

The enzyme displayed the highest level of affinity to methanol. FA and acetaldehyde were also substrates, which inhibited MDH at high concentrations. In this connection, for the selective detection of methanol with MDH biosensor the sensor response for FA must be suppressed. It is known that cyanide forms an adduct with PQQ, being a competitive inhibitor of the MDH when using methanol as a substrate. In the presence of 2 mM KCN the K_M values to methanol and formaldehyde were differenced significant – 0,23 mM and 7,2 mM, respectively.

The effect of cyanide concentration in the buffer solution on the MDH-sensor responses for methanol and formaldehyde were studied. With increasing cyanide concentrations up to 10 mM the inhibition level of MDH-biosensor responses for methanol and formaldehyde were differenced even more. There was practically complete inhibition of the sensor responses for FA while the oxidation activity of immobilized MDH towards methanol decreased slightly. As a result of cyanide effect selective detection of methanol in the presence of FA were allowed

The detection limit, linear range, sensitivity and stability of the mediator amperometric biosensors based on membrane fraction of M. dichloromethanicum for FA detection and MDH-sensor for methanol detection were determined and are listed in Table 1.

Table 1. Performance characteristics of amperomeric biosensors based on membrane fractions and MDH of methylobacteria.

	Membrane fraction of M. dichloromethanicum (FA)	МДГ M. nodulans (methanol)
Sensitivity coefficient, nA/mmol*L^-1	25±3	7200±300
Detection limit, mmol/L	0,05	0,0045
Upper limit of analytical range (KM), mmol	7,0±0,5	0,49±0,03
Relative standard deviation , (n=15), %	7,3	7,6
Long-term stability (10 days), %	63	12

The work was supported by the Federal Goal-oriented Program “Scientific and Scientific-Pedagogical Cadres of Innovative Russia” for 2009–2013, agreement № 14.B37.21.0561 and № 14.B37.21.1231.