Characterisation of the cold-active alcohol dehydrogenase in a new psychrotolerant bacterium from Antarctica

Psychrophyllic and psychrotolerant bacteria have successfully colonized polar and alpine regions and are able to grow efficiently at temperatures close to zero. At the enzymatic level, these organisms have to cope with reduction of chemical reaction rate induced by low temperatures in order to maintain adequate metabolic fluxes. To enable them to survive and grow in cold environments, psychrophyllic and psychrotolerant bacteria have evolved a complex range of adaptations to all of their cellular components, including their membranes. Among proteins, these are cold-active enzymes that differ from their mesophillic counterparts, and mainly in their thermal catalytic properties. The strain MS-5, isolated from Antarctic soil on the basis of its ability to grow on sodium dodecyl sulphate as a sole source of carbon and energy has been taxonomically classified as a psychrotolerant variant of Micrococcus luteus. This identity was confirmed by the presence of menaquinone-8 in the neutral lipid fraction of MS-5, 16S rDNA analysis, antibiotic susceptibility testing, analysis of bacterial lipids and biochemical tests. However, restriction fragment length polymorphism analysis of the inter-gene spacer region of the strain MS-5 and two mesophillic M.luteus strains suggested the strain MS-5 as being a novel strain of the normally mesophillic Micrococcus luteus. M. luteus strain MS-5 responds to the lowering the growth temperature like mesophillic strains of M. luteus by increasing the ratio of anteiso- to iso- branched fatty acids. Primary alcohol dehydrogenase (1 ADH), constitutively produced by M. luteus strain MS-5, is cold-active having a half-time of the enzyme activity at 60 C less than 2 minutes. Gel zymography procedure together with kinetic measurements showed the preference of the enzyme to medium-chain alcohols. According its N-terminal sequence and catalytic properties this ADH belongs among Group I NAD+- dependent ADHs. The enzyme has been purified to electrophoretic homogeneity by four-step purification procedure with 846-fold purification. The enzyme is a homotetramer of a subunit relative molecular mass of 45,000 as determined by size-exclusion chromatography and SDS-PAGE. Isoelectric point and pH optimum of the enzyme were 4.7 and 10.5 respectively. N-terminal sequence of the enzyme (S E K V R G V V A M A K D A P V T) has 64 percen t identity to ADH-E2 from Mycobacterium tuberculosis and ADH from Streptomyces coelicolor A3. Although a genomic library of M. luteus MS-5 constructed in lambda phage was found to be representative, attempts to clone the adh gene were unsuccessfulAvailable from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi