Zusammenfassung: Die NADP®-abhängige Aldehyd-Dehydrogenase von Acinetobacter calcoaceticus ist nach unseren früheren Befunden ein induzierbares Enzym aus intracytoplasmatischen Inclusionsmembranen. Wir gewannen es als Detergens/Phospholipid-Mischmicelle und demonstrieren in vorliegender Arbeit Ergebnisse zur kinetischen Charakterisierung dieses Enzyms. Die Aldehyd-Dehydrogenase hat ein pH-Optimum um pH 10 und wird durch Aldehydüber-schuß gehemmt. Der A^-Wert für NADP e liegt -unabhängig von der Kettenlänge der Aldehydsubstrate -bei 8.6 x IGT 5 M. Die K m -Werte für die Aldehyde fallen mit steigender Kettenlänge ab (von 1.6 l ( 3 beiButanal auf 1.5 10~6 bei Decanal). Auch die K { -Werte (für die Hemmung durch Aldehydüberschuß) sinken mit wachsender Kettenlänge von 7.5 10~3 (bei Butanal) auf 3.5 10~5M (bei Decanal) ab, genauso wie die Aldehyd konzentrationen, die jeweils die "optimale" Reaktionsgeschwindigkeit bedingen (bei Butanal 5mM, bei Decanal 6 ). Die Anzahl der inhibierenden Aldehydmoleküle pro Enzym-Substrat-Komplex beträgt n= 1. Das Enzym wird durch NADPH als Produkt gehemmt, nicht dagegen durch Fettsäuren. Eine Rückreaktion konnte nicht gemessen werden. Nicht-kompetitive Inhibitoren des Enzyms sind Sn 2 *, Fe 20 , Cu 2e , BOi e , CN 9 , EDTA, o-Phenanthrolin, p-Chlormercuribenzoat, Mercaptoethanol, Phenylmethylsulfonylfluorid und Diisopropylfluorophosphat, nicht dagegen lodacetat. Chloralhydrat ist ein kompetitiver Inhibitor gegen die Aldehyde. Aktivierend wirkte -abhängig von der Kettenlänge der AldehydeButansäure-ethylester.
Kinetics of membrane-bound aldehyde dehydrogenase of Acinetobacter calcoaceticusSummary: In recent investigations we were able to demonstrate that the NADP®-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too.The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The K s and K m values of the leading substrate NADP® were found to be 8.6 10~5 and 10.3 10~5M independent of the chain-length of the aldehydes. The K m values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 10~3, decanal: 1.5 10~6M).The K { values (for inhibition by aldehyde excess)
Purified aldehyde dehydrogenase (NADP+-dependent) of intracytoplasmic membranes of Acinetobacter cakcoaceticus could he incorporated from micelles formed during the purification procedure into liposomal membranes. Both the cholate dilution method and the ultrasonication method were suitable to produce enzyme liposomes. In unilamellar liposomes produced by phosphntidyl choline, the enzyme activity decreased to 1% (or less) of the original activity. In contrast, about 10% of the original activity could be preserved in unilamellar liposomes prepared from bacterinl phospholipids.The destruction of the enzyme liposomes induced by detergents (lauroyl sarcosinate) was followed by measuring the wavelength dependence of turbidity, which allowed us to draw conclusions on size and stability of the particles in the suspension. I n addition these measurements demonstrated that decanal and NADP+ did not destroy the liposomal structure a t concentrations necessary for the determination of enzyme activity.The liposomal enzyme was inactivated to a lesser degree by proteinase K than the micellar enzyme. Both phospholipase A, and D inactivated the enzyme incorporated into the liposomal membranes to about 50%. After t,reatment with phospholipase A,, the enzyme could be reactivated by bacterial phospholipids. After treatment with phospholipase D, no reactivation was possible by bacterial phospholipids.Die NADP-abhangige Aldehyddehydrogenase ist bei A . calcoaceticus ein Enzyin des Oxidations-Pathways der Alkane (AURICH u. EITNER 1977, SORGER u. AURICH 1978 und in den intracytoplasmatischen Umhiillungsmenibranen der Alkan-Inklusionen lokalisiert (VOAISEK et al. 1982). Sie liiljt sich durch Detergentien, wie Desoxycholat, Cholat, Laurylsarkosin und Triton X-100 solubilisieren (AURICH et al. 1985) und als Detergens-Phospholipid-Mischmizelle isolieren und reinigen . Wahrscheinlich ist Cardiolipin fur die Aktivitat des Enzynis essentiell (BERGMANN 1982
The growth conditions for the cultivation of Acinetobacter calcometicus in a commercial laboratory fermenter have been optimized. This was done taking former results of shake cultures as a basis. At the end of the logarithmic phase u p to 10 g/1 dry weight were obtained.The following culture conditions have been used: The medium contained 10 ml/l n-alkane and 6 g/l NH,CI. The pH was adjusted to 6.5 and PO, employed to 70% saturation. A 12 times higher amount of dry weight was observed compared with shake culture technique.
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