Purification and Characterization of aBacillus licheniformisPhosphatase Specific ford-α-Glycerophosphate

Bacillus licheniformis(“Fordʹs type”) was found to contain a novel enzyme,d-α-glycerophosphatase. The enzyme is highly specific ford-α-glycerophosphate, effecting little or no hydrolysis ofl-α- or β-glycerophosphate or other similar compounds. All other known α-glycerophosphatases preferentially hydrolyze thelisomer. The products of thed-α-glycerophosphatase reaction were identified as glycerol and inorganic phosphate. The enzyme is a monomer with an apparent molecular mass of approximately 25 kDa. As with most phosphatases, it requires divalent magnesium for activity, but unlike the nonspecific acid and alkaline phosphatases, its optimum pH is around neutral. ItsKmford-α-glycerophosphate in the presence of 1 mM Mg2+was found to be 4.3 mM.d-α-Glycerophosphatase was produced inB. licheniformisfermentations whether or not high levels of phosphate were present; the same was true of glycerol formation.d-α-Glycerophosphatase is not strongly inhibited by inorganic phosphate and would therefore be capable of catalyzing the formation of glycerol in the presence of high levels of phosphate. Thed-α-glycerophosphatase ofB. licheniformisis similar in characteristics tol-α-glycerophosphatases known to synthesize glycerolin vivo,suggesting thatd-α-glycerophosphatase may be the final enzyme in the fermentative glycerol formation pathway ofB. licheniformis.