Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration.
Polyphosphate kinase (ATP:polyphosphate phosphotransferase; EC 2.7.4.1), partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity. The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor. Chromatographic analyses of the product mixture produced from ADP and [32P]polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase (EC 3.6.1.11), adenosine triphosphatase (EC 3.6.1.4), and adenylate kinase (EC 2.7.4.3). Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C. It may be stored indefinitely as a lyophilized powder at -10 degrees C. Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme. A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium. The ease of purification and immobilization of E. coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration.
Hoffman
R C
RC
Department of Chemistry, Lebanon Valley College, Annville, Pennsylvania 17003.
Wyman
P L
PL
Smith
L E
LE
Nolt
C L
CL
Conley
J L
JL
Hevel
J M
JM
Warren
J P
JP
Reiner
G A
GA
Moe
O A
OA
D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000302:isolation & purification; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor)
D004800:Enzymes, Immobilized; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase