Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 was
increased with the addition of Pb, As, and Cu compared to the control with no heavy
metals. In addition, the presence of Ni, Cd, and Mn in the growth medium decreased
the level of ACC deaminase in all bacteria tested except for Klebsiella Mc173, and
the only bacterial strain that exhibited higher levels of activity in the presence of
these heavy metals was Serratia K120, which also promoted Helianthus annuus
plant growth in the presence of heavy metal contamination (Carlos et al. 2016).
19.5 Different Enzyme Assays
ACC deaminase enzyme activity may be assayed by monitoring the production of
either ammonia or α-ketobutyrate, the end products of the ACC deaminase catalyzed
reaction (Table 19.3). ACC deaminase is an inducible enzyme whose activity is
induced by the presence of its substrate, namely ACC at levels as low as 100 nM, and
the complete induction of the enzyme is a slow process that can take up to 10 h
(Glick 2004). The screening of microbes for the presence of this activity is started by
growing them in minimal media supplemented with ACC as the sole source of
nitrogen (Penrose and Glick 2003) under growth conditions suitable for the microbe
of interest. Briefly, ACC deaminase activity is measured in toluenized bacterial
extracts, which are then incubated with the substrate, ACC. The final reaction is
incubated with 2,4-dinitrophenylhydrazine that interacts with the newly released
α-ketobutyrate and, after the addition of NaOH, the absorbance is read at 540 nm.
The amount of α-ketobutyrate that is produced as a result of ACC deaminase activity
is determined using a standard curve ranging from 0 to 1.0 μmol of α-ketobutyrate.
Total protein is also estimated from the same toluenized bacterial extracts using
bovine serum albumin as a standard over the range of 0.05–1 mg. Finally, the
enzyme activity is determined in μmol of α-ketobutyrate produced per mg of total
protein present in the bacterial extract in 1 h (μmol mg
–1 h
–1 ). This method is
generally used for determining the ACC deaminase activity by non-rhizobial bacterial strains (Penrose and Glick 2003) whereas, a modified method is used for
Rhizobia (Duan et al. 2009). The above-mentioned enzymatic assay can precisely
detect 0.1 μmol of α-ketobutyrate produced in the reaction. Nevertheless, a low level
of ACC deaminase activity (! 20 nmol mg
–1 h
–1 ) is required to allow bacteria to
grow on ACC and to lower stress ethylene levels. Interestingly, bacteria with higher
enzymatic activity do not necessarily perform any better than the organisms that
possess lower levels (<20 nmol mg
–1 h
–1 ) of enzyme activity (Penrose and Glick
2003).
The ability of bacterial strains that produce ACC deaminase to consume ACC as a
sole source of nitrogen has also been documented (Li et al. 2011). Nevertheless,
identification of bacteria merely by growing them on minimal media containing
ACC as the sole nitrogen source can overestimate the ACC deaminase prevalence in
any environmental niche since nitrogen-fixing bacteria that do not contain ACC
deaminase may also grow on this medium. In addition, when the presence of the
374
S. Ali and B. R. Glick
increased with the addition of Pb, As, and Cu compared to the control with no heavy
metals. In addition, the presence of Ni, Cd, and Mn in the growth medium decreased
the level of ACC deaminase in all bacteria tested except for Klebsiella Mc173, and
the only bacterial strain that exhibited higher levels of activity in the presence of
these heavy metals was Serratia K120, which also promoted Helianthus annuus
plant growth in the presence of heavy metal contamination (Carlos et al. 2016).
19.5 Different Enzyme Assays
ACC deaminase enzyme activity may be assayed by monitoring the production of
either ammonia or α-ketobutyrate, the end products of the ACC deaminase catalyzed
reaction (Table 19.3). ACC deaminase is an inducible enzyme whose activity is
induced by the presence of its substrate, namely ACC at levels as low as 100 nM, and
the complete induction of the enzyme is a slow process that can take up to 10 h
(Glick 2004). The screening of microbes for the presence of this activity is started by
growing them in minimal media supplemented with ACC as the sole source of
nitrogen (Penrose and Glick 2003) under growth conditions suitable for the microbe
of interest. Briefly, ACC deaminase activity is measured in toluenized bacterial
extracts, which are then incubated with the substrate, ACC. The final reaction is
incubated with 2,4-dinitrophenylhydrazine that interacts with the newly released
α-ketobutyrate and, after the addition of NaOH, the absorbance is read at 540 nm.
The amount of α-ketobutyrate that is produced as a result of ACC deaminase activity
is determined using a standard curve ranging from 0 to 1.0 μmol of α-ketobutyrate.
Total protein is also estimated from the same toluenized bacterial extracts using
bovine serum albumin as a standard over the range of 0.05–1 mg. Finally, the
enzyme activity is determined in μmol of α-ketobutyrate produced per mg of total
protein present in the bacterial extract in 1 h (μmol mg
–1 h
–1 ). This method is
generally used for determining the ACC deaminase activity by non-rhizobial bacterial strains (Penrose and Glick 2003) whereas, a modified method is used for
Rhizobia (Duan et al. 2009). The above-mentioned enzymatic assay can precisely
detect 0.1 μmol of α-ketobutyrate produced in the reaction. Nevertheless, a low level
of ACC deaminase activity (! 20 nmol mg
–1 h
–1 ) is required to allow bacteria to
grow on ACC and to lower stress ethylene levels. Interestingly, bacteria with higher
enzymatic activity do not necessarily perform any better than the organisms that
possess lower levels (<20 nmol mg
–1 h
–1 ) of enzyme activity (Penrose and Glick
2003).
The ability of bacterial strains that produce ACC deaminase to consume ACC as a
sole source of nitrogen has also been documented (Li et al. 2011). Nevertheless,
identification of bacteria merely by growing them on minimal media containing
ACC as the sole nitrogen source can overestimate the ACC deaminase prevalence in
any environmental niche since nitrogen-fixing bacteria that do not contain ACC
deaminase may also grow on this medium. In addition, when the presence of the
374
S. Ali and B. R. Glick