Enzymes Active in the Intact Spore 



Norman L. Lawrence^ 



New York State Agricultural Experiment Station, 

 Cornell University, Geneva, New York 



IN WORKING with spore enzymes, one of the requirements is clean spore 

 suspensions. Unfortunately there is no accepted criterion of cleanliness. 

 However, it is generally recognized that a large number of washings with 

 water, buffers or acids are necessary to eliminate contaminating material 

 from the medium or debris from the sporangium, and that care must be 

 exercised to prevent germination and destruction of enzymes that may be 

 present in the spore proper. 



In at least four cases, studies with intact spores have demonstrated enzy- 

 matic activity which does not appear to be attributable to vegetative residue 

 or to the presence of germinated spores. These enzymes are: (1) alanine 

 racemase, (2) catalase, (3) adenosine deaminase, and (4) nucleoside ribo- 

 sidase. In addition, there are indications of other active systems which I 

 hope will be discussed by the workers involved. 



Alanine racemase. The report of Stewart and Halvorson (1953) on alanine 

 racemase was the first clear-cut demonstration of an active enzyme in 

 bacterial spores. Resting spores of Bacillus terminalis (a strain of B. cereus) 

 formed a maximum of 140 micromoles of D-alanine from the L-isomer pei 

 hour per milligram at pH 8.5. Eight strains of seven species of Bacillus 

 were tested and activity was found in all strains, ranging from a Q of 10 

 to a Q of 84 ^M/hr/mg. The enzyme was specific for alanine, and in another 

 paper by the same authors (1954) active extracts were shown to require 

 pyridoxal phosphate. It was further shown that the enzyme in whole spores 

 and in particulate fragments was stable at 80°C for 2 hours, and resistant to 

 pepsin digestion. By sonic vibration and differential centrifugation, a 

 "soluble", heat- and pepsin-sensitive enzyme was obtained from the spores. 

 The implication here is that combination of certain groups of the enzyme 

 with groups of the larger particles of a certain minimum size results in a 

 stable but active enzyme. I wonder whether dipicolinic acid plays any part 

 here. 



In a later paper. Church and others (1954) demonstrated that the activity 

 of this enzyme is not essential for germination. B. globigii spores, which 



^Approved by the Director of the New York State Agricultural Experiment Station 

 for publication as Journal Paper No. 1092, Sept. 16, 1957. 



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