IGEM:IMPERIAL/2009/Assays Protocols/Trehalose/OtsA

OtsA : Trehalose –6-Phosphate synthase
TPS Assay : A quantitative assay of the activity of Trehalose –6-Phosphate synthase (units of activity given in μmol of product per minute).

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UDPG G-6-P Tris-HCl Buffer MnCl2 heparin salt HCl NaOH anthrone Shodex Sugar

Alternative Assay
Link to website for assay

Trehalose –6-Phosphate synthase assay : non-radioative method Adapted from Vandercammen et al (Eur. J. Biochem. 182, 613-620., 1989)

Reaction:

Tre6Psynthase:

UDPglucose + Glucose-6-phosphate  =>       Trehalose-6P + UDP

Enzymatic coupling:

Pyruvate kinase:                   UDP + PEP     =>       Pyruvate + UTP

Lactate Dehydrogenase:       Pyruvate + NADH =>             Lactate + NAD

Preparation of crude extract:

- Break open 15-20 mg dry mass (109 cells) in 0.5 ml extraction buffer (Hepes 20 mM, pH 7.1, DTT 1mM, KCl 100 mM; cocktail inhibitor from Boerhinger)/ 1 g glass beads by vortexing 6 times 30 sec with 30 sec interval in ice.

- Centrifuge tubes at 3000g for 5 min at 4°C

- transfer supernatant into eppendorf tube

- Centrifuge 15min in eppendorf centrifuge at full speed at 4°C

Enzymatic assay:

In 1 ml final volume

Mixture TPS                                                                                                 0.5 ml

(Hepes 50 mM, pH 7.1, KCl 100 mM; EDTA 2mM, MgSO4 5 mM)

UDPglucose 20 mM                                                                                     0.1 ml

Glc-6-P/ F-6-P 100 mM/30 mM                                                                  0.1 ml

Water                                                                                                           0.2 ml

Sample                                                                                              0.1 ml

Incubate at 42°C

After 10, 20, 30 min

Take 0.25 ml in 0.75 ml of ice-cold UDP mixture kept in ice-cold water bath

Measure OD340        (E0)

Add Lactate dehydrogenase 100 U/ml            1 µl

Read OD340              (E1)

Add Pyruvate kinase   100U/ml                      1 µl

Read OD340              (E2)

Calcul of activity:

Tre6PS activity (in nmol UDP produced/ min / mg protein) is given by:

D (Es2-Es1) – (D Ebk2-Ebk1)/ 6.22 x 1000/100 x 1000/4 x 1/time x 1/protein (mg/ml)

With E is value of OD for the sample and Ebk value OD for the blank

The blank is carried out with the extract that has been heat inactivated for 5 min at 80°C before addition to the mixture.