Corynebacterium glutamicum - JUWEL - Forschungszentrum Jülich

5.1. Material and Methods
concentration was in the proper range of 0.1 to 6 g/L. In the used test strips, the glucose
is converted to gluconolacton by glucoseoxidase. During this reaction, a small current is
produced which is measured.
After a finished experiment, the glucose concentrations in the samples of supernatant
were measured by an enzymatic assay in 96 well microtiter plates using hexokinase
and glucose-6-phoshatedehydrogenase, similar to the Boehringer enzymatic analysis kit.
The samples were diluted in water to about 0.3 g/L glucose. Standard solutions of
0.05-0.5 g/L glucose were measured at least in duplo on each plate. All samples were
measured in duplo or triplo. In the assay, the glucose is converted to glucose-6-phosphate
by hexokinase using ATP. This glucose-6-phosphate is then converted together with NAD
to 6-phosphogluconate and NADH by glucose-6-phoshatedehydrogenase. The NADH
was measured spectrophotometrically at 340 nm in a Thermomax microtiterplate-reader
(Molecular Devices, Sunnyvale Ca., USA).
The enzymatic method in microtiter plates is more accurate and less expensive than
the measurement in the teststrips mentioned above, but it takes much more time.
Amino Acids
The concentrations of several amino acids were determined in the stored samples of
the supernatant by reversed phase high-performance liquid chromatography (HPLC,
Sykam, Gilching, Germany) after derivatisation with ortho-phthaldialdehyde (OPA)
(Lindroth and Mopper, 1979). The amino acids of interest were L-alanine, L-glycine,
L-valine, L-isoleucine and L-leucine. Samples were first diluted with water in order to
contain 10-200 µM of the relevant amino acids. The used standards contained 50 µM of
the amino acids and 500 µM ammonium. A typical chromatogram of standard solutions
is shown in figure 5.4.
The amino acids were derivatised with OPA and mercaptoethanol (OPA reagent,
Pierce Europe BV, Oud-Beijerland, the Netherlands) for 1.5 minutes before they were
separated on a reversed-phase column (Lichrospher 100 RP 18-5, 125 mm long, 4 mm
diameter and 100 ˚A pore size, Merck, Darmstadt, Germany). During this pre-column
derivatisation, the primary amines react with OPA and mercaptoethanol to the corresponding
fluorescent isoindols. The elution from the column is done isocratically with
a filtered solution containing 50% v/v of a 10 mM phosphate puffer of pH 7.2, 35%
v/v methanol and 15% v/v acetonitril. Behind the column, the isoindols are detected
fluorimetrically (Shimadzu RF-535 detector, excitation wavelength 330 nm, emission
wavelength 450 nm, Shimadzu, Duisburg, Germany)
The isocratic elution with the used elution buffer at 0.9 mL/min, was found to be
suitable for the fast separation of the relevant amino acids. Especially the separation of
ammonium and L-valine was found to be rather difficult with the used system, which
was originally used with a gradient with an increasing methanol concentration. This
separation is very important for our purpose, because high concentrations of ammo-
99