plant surface microbiology.pdf

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Absorbance at 540 nm
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
Donna M. Penrose and Bernard R. Glick
0
0 0.2 0.4 0.6 0.8 1
-ketobutyrate, moles
to use, the stock solution is diluted with the same buffer to make a 10-mM
solution from which a standard concentration curve is generated. Each in a
series of known a-ketobutyrate concentrations is prepared in a volume of
200 ml and transferred to a glass test tube (100x13 mm); each point in the
series is assayed in duplicate. Three hundred ml of the 2,4-dinitrophenylhydrazine
reagent (0.2 % 2,4-dinitrophenyl-hydrazine in 2 N HCl; Sigma-
Aldrich Co.) is added to each glass tube and the contents are vortexed and
incubated at 30 °C for 30 min during which time the a-ketobutyrate is derivatized
as a phenylhydrazone. The color of the phenylhydrazone is developed by
the addition of 2.0 ml of 2 N NaOH; after mixing, the absorbance of the mixture
is measured at 540 nm.
5.1 Assay of ACC Deaminase Activity in Bacterial Extracts
Fig. 1. Standard curve of
a-ketobutyrate versus
absorbance at 540 nm
5.1.1 Preparation of Bacterial Extracts
ACC deaminase activity is measured in bacterial extracts prepared in the following
manner. Bacterial cell pellets, prepared as described in Section 3, are
each suspended in 1 ml of 0.1 M Tris-HCl, pH 7.6 and transferred to a 1.5-ml
microcentrifuge tube. The contents of the 1.5-ml microcentrifuge tube are
centrifuged at 16,000xg for 5 min in a Brinkmann microcentrifuge and the
supernatant is removed with a fine-tip transfer pipette. The pellet is suspended
in 600 ml of 0.1 M Tris-HCl, pH 8.5. Thirty ml of toluene is added to the
cell suspension and vortexed at the highest setting for 30 s.At this point, a 100ml
aliquot of the “toluenized cells” is set aside and stored at 4 °C for protein
assay at a later time. The remaining toluenized cell suspension is immediately
assayed for ACC deaminase activity.