Premium
Multiple immobilized enzyme reactors: Determination of pyruvate and phosphoenolpyruvate concentrations using immobilized lactate dehydrogenase and pyruvate kinase
Author(s) -
Newirth T. L.,
Diegelman M. A.,
Pye E. K.,
Kallen Roland G.
Publication year - 1973
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260150608
Subject(s) - pyruvate kinase , lactate dehydrogenase , phosphoenolpyruvate carboxykinase , chemistry , pyruvate dehydrogenase kinase , enzyme , chromatography , nicotinamide adenine dinucleotide , pyruvate dehydrogenase phosphatase , immobilized enzyme , biochemistry , pyruvate decarboxylation , dehydrogenase , pyruvate dehydrogenase complex , nad+ kinase , glycolysis
Lactate dehydrogenase (LDH) and pyruvate kinase (PK), immobilized on solid glass beads by diazotization, were used in packed bed reactors to analyze for both pyruvate (PYR) and phosphoenolpyruvate (PEP) through the disappearance of β‐nicotinamide adenine dinucleotide (NADH) monitored spectrophotometrically at 340 nm. Packed bed reactors containing PK and/or LDH were also capable of monitoring continuously varying concentrations of adenosine‐5′‐diphosphate (ADP), PEP, and PYR. The immobilized enzymes (∼40 μg/g glass) retained about 4% of the activity of the soluble enzymes. Preparations of immobilized LDH and PK exhibited enhanced stability when maintained in the presence of β‐mercaptoethanol and NADH or EDTA, respectively, and were shown to regain 75% of the original activity after four months storage at 4°C.