BUTYRIC ACID-BUTANOL FERMENTATIONS 47 



acetyl-SCoA in the absence of free coenzyme A (reactions 

 VI, V, and IV) has been demonstrated by spectrophoto- 

 metric methods. 36 - 53 When free coenzyme A was added to 

 such a reaction mixture, the accumulated acetoacetyl-SCoA 

 was rapidly converted to acetyl-SCoA by reaction III. 

 Unfractionated CI. kluyveri extracts were found by Bartsch 38 

 to possess acetoacetyl thiolase activity corresponding to the 

 decomposition of approximately 2 micromoles of acetoacetyl- 

 SCoA per minute per milligram of protein. /?-hydroxy- 

 butyryl-SCoA dehydrogenase activity (reaction IV) has 

 also been demonstrated qualitatively by Bartsch by using 

 a DPNH-generating system to reduce the N-acetyl thioeth- 

 anolamine analogue of acetoacetyl-SCoA. The reaction was 

 followed by the decrease in ultraviolet absorption of the 

 analogue at 303 m^. The reduction product has not been 

 characterized in this system. The corresponding enzyme 

 from rat liver mitochondria has been shown to produce 

 l ( + ) -/?-hydroxybutyryl-SCoA. 54 ' 55 



Crotonase (reaction V) activity is very high in CI. kluy- 

 veri extracts. 38 Using crotonyl-SCoA as a substrate and 

 following the hydration of the double bond by means of its 

 ultraviolet absorption at 263 m^,, the crotonase activity was 

 shown to correspond to the utilization of 570 micromoles 

 of crotonyl-SCoA per minute per milligram of crude 

 protein. This is roughly 20 times the specific activity of 

 ox liver extracts, a rich animal source of the enzyme. 40 



Butyryl-SCoA dehydrogenase activity (reaction VI) has 

 been observed by coupling the oxidation of the pantotheine 

 analogue of butyryl-SCoA with the reduction of indo- 

 phenol, 38 and by coupling the reduction of crotonyl cysta- 

 mine with the oxidation of leucosafranine. 56 By means of 

 the latter technique it was found that crude extracts of 

 CI. kluyveri were twice as active with respect to butyryl- 

 SCoA dehydrogenase as the corresponding animal enzyme 



