Aldolase, from Clostridium perfringens

TV-Acetyl neuraminic acid aldolase [from Clostridium perfringens, TV-acetyIneuraminic acid pyruvate lyase] [9027-60-5] 32,000 [EC 4.1.3.3]. Purified by extraction with H2O,... 

Commercial A -acetylneuraminic acid aldolase from Clostridium perfringens (NeuAcA EC 4.1.3.3) catalyzes the addition of pyruvate to A-acetyl-D-mannosamine. A number of sialic acid related carbohydrates are obtained with the natural substrate22"24 or via replacement by aldose derivatives containing modifications at positions C-2, -4, or -6 (Table 4)22,23,25 26. Generally, a high level of asymmetric induction is retained, with the exception of D-arabinose (epimeric at C-3) where stereorandom product formation occurs 25 2t The unfavorable equilibrium constant requires that the reaction must be driven forward by using an excess of one of the components in order to achieve satisfactory conversion (preferably 7-10 equivalents of pyruvate, for economic reasons). 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

A-Acetylneuraminic acid aldolase (NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate to A-acetyl-D-mannosamine (1) to form the parent sialic acid (3) (Fig. 4). The NeuA lyases found in both bacteria and animals are type I enzymes that form an enamine intermediate with pyruvate and promote a j/-face attack to the aldehyde carbonyl group with formation of a (4S) configurated stereo center. Enzyme preparations from Clostridium perfringens and E. coli are commercially available, and the latter enzyme has been cloned, overexpressed [44,45], and its three-dimensional structure determined [46]. The enzyme has a broad pH optimum around 7.5 and is quite stable in solution at ambient temperature [47]. 

---