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Alcohol dehydrogenases (Oxidases) from Methylobacterium extorquens and strawberry Fragaria x ananassa (cv Elsanta). Isolation, study, expression and comparison

By Παναγιώτης Κουτσομπόγερας and Παναγιώτης Ξενοφών Κουτσομπόγερας

Abstract

The individual, as well as the combinational activity of alcohol dehydrogenases (ADH) - from bacterium Methylobacterium extorquens (with CABI number of registration IMI369321) and from strawberry Fragaria x ananassa cv. Elsanta - were studied in this work. Additionally there was a study of the production of furaneol (2,5-dimethyl-4-hydroxy-2H-furan-3-one or DMHF) by these enzyme sources, from various substrates. The optimal pH of the enzyme action was found to be pH 6,0 for the bacterium while for the seeds of strawberry was pH 10,0 respectively. In cell free extracts of M. extorquens - an organism capable of growing in 1,2-propanediol as source of energy and carbon - high activities of NAD-dependent ADH were found with 1,2-propanediol and methanol as substrates. The best substrates for the ADH from the body of strawberry were 1-propanol and ethanol. Respectively the best substrates for ADH from the seeds of strawberry (for pH 6,0) were ethanol and methanol. Similarly the best substrates for the ADH from the seeds of strawberry (for pH 10,0) were found to be ethanol and methanol. No important ADH activity was observed for aldehydes with the 2 enzymes of strawberry, while important activity was observed for the ADH from the bacterium. From the presented elements it is obvious that certain heavy metals (Pb, Cr, Ba), as well as the use of EDTA and NaN3 decrease the enzyme activity, for both the bacterium and the strawberry. Apart from the comparisons for the substrate, the ADH from the bacterium was cleaned 2,8 times while ADH from strawberry was cleaned 2,3 times respectively. The corresponding size of cleaning for the enzyme of the seeds of was 2,5. ADH enzyme activity was also observed after native electrophoresis and the export of protein zones. It is also reported here that the molecular weight of enzyme from the seeds of strawberry is 47,0 kDa while from the body of strawberry is 24,6 kDa. Respectively for the Methylobacterium extorquens the molecular weight is 45,1 kDa similar enough with the one from the seeds of strawberry. The results from the HPLC analysis showed that the best precursor molecules for the production of DMHF in the combined mixture [bacterium & body of strawberry ], comparatively with the enzyme extract of the body of strawberry, were found to be 1-propanol, 1,2-propanediol, fructose, glucose and mannitol. The best precursor molecules for the production of DMHF in the combined mix [bacterium & seeds of strawberry] - comparatively with the enzyme extract of the seeds of strawberry, were found to be 1-propanol, ramnose , sucrose, fructose and glucose Moreover a comparison of the oxidative capacities of the corresponding enzymes (ADH) regarding the bacterium and the strawberry revealed that the combination of their activities produced higher activity from the theoretical sum from their individual one. All of the above leads us to the conclusion that the combined mixture of the enzymes from the three different sources reacts at a higher percentage when they are present all together and thus strengthens the metabolic pathway for synthesizing odour compounds such as DMHF. In addition ADHs from the bacterium and the strawberry tend to have more efficient collaboration when these two are simultaneously present. The same conclusion is in effect for the ADH from the seeds from strawberries and the strawberry. On the other hand the collaboration of ADH from the bacterium and the seeds from strawberries is lessened when the two sources of enzymes are present together. With regard to the thermodynamic results that are derived from Arrhenius equation having 1,2-propanediol - as substrate for the enzyme - the following results were obtained. The activation energy (Ea) of the enzyme from the seeds of strawberry corresponds to the 85% of the activation energy of the corresponding enzyme from the strawberry while the corresponding Ea of the enzyme from the Methylobacterium extorquens is roughly the same with the enzyme from the strawberry. Moreover the change of enthalpy (ΔH) for the enzyme from the seeds of strawberry corresponds to the 84% of ΔH the corresponding enzyme from the strawberry while the corresponding ΔH of the enzyme from Methylobacterium extorquens is roughly the same with the ΔH of the enzyme from strawberry. Respectively the change of free energy (ΔG) for the enzyme from the seeds of strawberry corresponds to 87 % of the corresponding enzyme from the strawberry while the corresponding ΔG of the enzyme from Methylobacterium extorquens is roughly the same with the ΔG of the enzyme from strawberry.

Topics: Αλκοολικές δεϋδρογονάσες, Μεθυλοβακτήρια, Φράουλες, Alcohol dehydrogenase, Methylobacteria, Strawberry
Publisher: National and Kapodistrian University of Athens
Year: 2007
DOI identifier: 10.12681/eadd/18498
OAI identifier:
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