Neutral Trehalase Assay
This method is modified from Kopp, Muller and Holzer (1993). Molecular Analysis
of the Neutral Trehalase Gene from Saccharomyces cerevisiae.
(1) Grow the plates upside down with the Whatman paper facing down.
(2) Place the filter paper in chloroform for 5 minutes, upside down.
(3) Remove the chloroform and allow the filter to air until there is no more
moisture visible on the surface, this takes approximately 30 minutes.
(4) Incubate the filter paper in buffer for 20 minutes. The buffer is made from
3.6mM ATP, 0.18mM cAMP and 3.6mM MgCl2 dissolved in 50mM
imidazole-HCl that has been adjusted to pH7. Approximately 20ml is needed
per plate, strain.
Therefore in 80ml of buffer 0.159g of ATP is needed, 0.005g of cAMP and
0.059g of MgCl2. 100ml of Imidazole-HCl is made from 0.523g of powder.
(5) During this incubation the overlay mixture is prepared, each plate needs 10ml
of assay mixture. For 20ml add 3.78g trehalose and 0.2g of agarose to 19ml of
Imidazole-HCl, this solution is melted in a microwave and then placed at
50oC. Immediately before use this solution is added to 1mg N-ethylmalemide
(Sigma), 197 units of horse radish peroxidase (HRP, Sigma), 160 units of
glucose oxidase (Sigma, G9010) and 9.6mg o-Dianisidine Dihydrochloride
(Sigma, D9154).
The o-dianisidine comes in 10mg tablets, before this stage is reached dissolve one
tablet in 1ml SDW and use the whole of the resulting solution. Glucose oxidase
comes at 10,000 units per 6ml and therefore 96 l is required.
(5) Remove the excess buffer and immediately pour over the assay mixture as
soon as it has mixed. Leave to incubate in the CT room. After approximately 20
hours clear brown rings of activity will be present around the filter papers.
THEORY:
The chloroform allows permealisation of the cell walls, allowing the substrates to
react. The buffer ensures that neutral trehalase enzymes are activated. The trehalose in
the overlay mixture is hydrolysed to glucose by the trehalase enzymes. Then,
glucose  gluconic acid + H2O2 at pH7 via glucose oxidase
H2O2 + o-diansidine  colour change catalysed by HRP
o-diansidine is a chromogen and a peroxidase substrate that results in a yellow-orange
end product. The HRP and H2O2 complex oxidises the chromogen.
The N-ethylmalemide reacts with thiol groups and is therefore thought to be included
as a protease inhibitor, therefore, protecting the enzymes involved in the reaction or it
may inactivate enzymes such as catalase which may compete for the H2O2.