Thermoanaerobacter alcohol dehydrogenases revisited

Abstract

Chirality – the „handedness“ of life – and its influence on living organisms has given rise to and fueled the field of asymmetric synthesis. At the intersection of biology and chemistry, the search for biological agents capable of assisting in the resolution of racemic compounds and their formation from pro-chiral starting materials has led to the characterization of a number of robust catalysts including alcohol dehydrogenases (ADHs). Due to their high thermostability and solvent tolerance, ADHs from Thermoanaerobacter spp. have received particular attention since the 1980s, most notably T. pseudethanolicus. However, as most members of the genus remain unexplored in terms of their ADH activities and many have not been whole genome sequenced, a culture-based approach was undertaken in the study herein to elucidate the ADH activities of the whole genus by substrate mapping. A handful of Thermoanaerobacter strains showed NADP+-dependent ADH activities on secondary alcohols similar to that of T. pseudethanolicus, with crude ADH extracts reaching activities of over 30 mU/mg protein. Secondary to primary ADH activity ratio (SADH/PADH) remained virtually unchanged for most members of the genus at temperatures below their Topt. Notable exceptions included T. pseudethanolicus which showed a higher SADH/PADH activity ratio when cultivated at 50°C while T. italicus, T. pentosaceus, T. siderophilus, and C. subterraneus subsp. tengcongensis showed a higher SADH/PADH when cultivated at their Topt. Growth media supplementation with peptone and C2-4 alcohols resulted in multifold increase in ADH activities and overall better growth of T. brockii subsp. finnii, T. pseudethanolicus, and T. sulfurigignens whose whole-cells were used to catalyze oxidations and reductions of chiral secondary alcohols, diols and pro-chiral ketones respectively. The SADH/PADH ratio was frequently increased by low concentrations of 1-propanol. While reaction products could not be evaluated in terms of optical purity, results warrant further investigation as all substrates were acted upon.

Hendni (e. chirality) og áhrif hennar á lífverur hefur drifið áfram þróun ósamhverfra efnafræðiferla. Leit að lífefnum sem geta haft milligöngu um aðgreiningu handhverfra efnablanda sem og myndun þeirra hefur leitt til uppgötvunar á fjölda harðgerra hvata, þ.á.m. alkóhóldehýdrógenasa (ADH). Þökk sé hitastöðugleika þeirra og leysiefnaþols hafa ADH úr dreifkjörnungaættkvíslinni Thermoanaerobacter vakið athygli vísindasamfélagsins, sérstaklega dehýdrógenasar úr tegundinni T. pseudethanolicus. Þar sem meirihluti ættkvíslarinnar hefur ekki verið rannsakaður m.t.t. alkóhóldehýdrógenasavirkni og fáir meðlimir hennar hafa verið heilraðgreindir var ákveðið að kortleggja hvarfefnabreidd ættkvíslarinnar. Margir stofnanna reyndust virkir á hendnum alkóhólum líkt og T. pseudethanolicus og ensímvirkni var mest 30 mU/mg prótein. SADH/PADH virknihlutfallið reyndist óbreytt fyrir flesta meðlimi ættkvíslarinnar við Topt að undanskildum T. pseudethanolicus, T. italicus, T. pentosaceus, and C. subterraneus subsp. tengcongensis. Þegar peptóni og stuttum alkóhólum var í bætt við fljótandi ræktunaræti jókst ADH virkni og vöxtur T. brockii subsp. finnii, T. pseudethanolicus og T. sulfurigignens sem síðar voru notaðir til heilfrumuoxunar og afoxunar á hendnum samböndum. SADH/PADH virknihlutfallið jókst í mörgum tilfellum í kjölfar 1-própanól viðbótar. Niðurstöður gefa tilefni til nánari rannsókna, þó svo að ekki hafi verið hægt að greina ljósvirkni myndunarefna, þar sem bæði díól voru oxuð og greinótt ketónsambönd afoxuð.