Abstract
Erythritol is a four carbon polyol that is roughly 70% as sweet as sucrose and is not metabolized
by the human body. It can therefore be used in low-calorie products and as sweetener for
diabetic and obesitas diets. Besides sweetening foodstuffs, this compound can also be used as a
precursor for a wide range of products and applications. An usefull and widely applied method
of measuring erythritol concentrations already exists, which uses HPLC. This approach cannot
provide accurate measurements when the concentrations of the samples are below the detection
threshold of HPLC. A new, recent imagined method, uses a coupled enzymatic assay as an
analytical tool to measure erythritol concentrations. The range of measurable concentrations in
these assays, is thought to go below the threshold of HPLC. Bacillus abortus is able to metabolize
erythritol, and contains four genes for the metabolism of erythritol in an inducible operon. The
first gene encodes for an erythritol kinase (eryA) and has already been succesful cloned into E.
coli. The aim of this project was to clone, overexpress and purify the EryA enzyme and use it for
a coupled enzymatic assay for erythritol. Towards this, the eryA gene was synthesized and
initially subcloned into the pQE30 expression vector. The expression of the EryA protein was
analysed from this construct in both the XL-1Blue strain (no regulation of expression) and the
M15 prep4+ strain (no expression before induction). Even though the protein was expressed in
both conditions, the protein was always found to be in the insoluble pellet fraction. The eryA
gene was then subcloned into the pET28b vector and transformed into BL21DE3 cells and
expression of EryA protein was analysed. Again, the protein was found in the insoluble fraction.
To overcome the problem of insoluble protein, we switched to auto-inducing media and tested
both the pQE30 and pET28b constructs under these conditions. In all the cases, though the
protein was optimally expressed, it was still found to be in the insoluble fraction.