Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that
catalyzes the conversion of glucose-6-phosphate to
6-phosphoglucolactone with the reduction of NADP+ to NADPH. It is
the first component of the pentose phosphate pathway (PPP) that
is a central and important biochemical pathway. The PPP functions
to produce building block compounds and intermediates for energy
production. The first three enzymes of the PPP are referred to as
the oxidative phase and produce NADPH that is crucial for dealing
with oxygen stress, especially in people with G6PD deficiency.
There are tests for G6PD deficiency, but those used in the field
are not reliable. The existing assays for G6PD use the NADPH it
generates to reduce a dye and produce a colour change, which can
be observed with the naked eye and can be used to measure the
kinetic parameters of the purified G6PD in the laboratory.
Unfortunately, it suffers from a number of problems when used
with crude preparations under field conditions. The equilibrium
constant for the G6PD reaction is close to 1 so that the G6PD
substrate will only be depleted if subsequent enzymes in the PPP
are present. The extent of colour change will also depend upon
the activity of the next two enzymes in the PPP and these may
vary from person to person. The mutant forms of G6PD may be
unstable, and may gives rise to false positive results during the
assays. Hence, sensitive and reliable assays are needed.
Here, we report an improvised colorimetric method for G6PD
detection using tetrazolium salt, WST-8. The method used
enzyme-coupling reaction of G6PD with another two downstream
enzymes in the PPP. Addition of large excess of the two enzymes
decreased the response time and increases the sensitivity of the
test. Consequently, this enables detection of G6PD enzyme
activity at low substrate concentrations compared with an assay
of NADPH absorbance at 340 nm. The methods also generate NADPH
eight times faster than reaction with G6PD alone. This makes the
test more sensitive and predictive, which is also ideal for
industrial applications.
We also extend the application of the colorimetric WST-8 assay
for screening of recombinant libraries in a directed evolution
study for improved stability. The screening protocols were
modified to colony agar-blotting using filter paper as primary
screening and 96 well plates for secondary screening. The orange
coloured formazans produced by the reaction, make it suitable and
reliable for high-throughput qualitative and quantitative assays
for dehydrogenases. With only four rounds of evolution, the
method successfully identified promising variants with far
improved thermal and chemical (urea) stability.
Studies characterizing the properties and understanding the
effect of mutations on variants are also discussed. The mutations
responsible for improved stability were found located at the
large structural NADP+ domain that is important for integrity and
stability of the structure. The results also showed that the
mutations brought changes in the oligomeric forms of G6PD. The
thermostable variants were found to form higher order oligomers
compared to the native enzyme. The correlation between enzyme
stability and oligomerisation was also studied briefly
