Application of Enzyme Activity Probes to Characterize Aerobic Microorganisms

25 Pages·2007·0.73 MB·English

by  Anna Hoover

Application of Enzyme Activity Probes to
Characterize Aerobic Microorganisms
in Groundwater at PGDP, INL, & elsewhere
M. Hope Lee
Biodegradation / Bioremediation of TCE
TCE can be degraded by bacteria
•
Several different mechanisms, including two primary ones:
•
1. Anaerobic reductive dechlorination (ARD)
2. Aerobic cometabolic oxidation
Bioremediation technology can be based on microbial
•
degradation capacity
Tools are needed to detect appropriate enzyme systems and
•
assess their activity in the environment
Molecular Data: What do they tell you?
Do any bacteria in the sample carry the genes for contaminant
(TCE) degradation?
(DNA Probe, e.g. QPCR)
YES NO
Are the degradative genes expressed?
No
(mRNA Probe) On-going TCE
degradation
YES NO
Are the TCE-degrading enzymes active? No
(Enzyme Activity Probe) On-going TCE
degradation
YES NO
No
TCE
On-going TCE
degradation
degradation
Microbial metabolism, cometabolism,
and enzyme activity probes
fluorogenic
TCE methane O
2
substrate
Methane
Monooxygenase
* * *
Cometa- Energy
e- Aerobic
bolism production
respiration
fluorescent oxidized
TCE
product epoxide product H O
2
*
Other enzymes include propane, ammonium, toluene oxygenases, etc.
What is an Enzyme Activity Probe?
Methane
Toluene
Probe
TCE
Cell Labeled cell
Other natural substrates that support TCE oxidation:
benzene, ammonia, phenol
Representative Enzyme Probes and Target Systems:
N
CH
CH C
3 C
toluene phenylacetylene trans-cinnamonitrile
(natural (2,3-TDO) (2,3-TDO)
substrate)
(2-TMO, 3-TMO)
O O
O
CH
OH
C
coumarin
(sMMO)
OH C
CH
3-hydroxy-phenylacetylene 3-ethynyl-benzoate
(2-TMO) (side chain MO)
( 3-TMO)
Results of applying Enzyme Activity Probes to
groundwater
Negative probe response
Total bacterial cell count Positive probe response
Control Assays
Purpose: To ensure that the measured degradation is attributable to the organisms
of interest; verify that either the sMMO or toluene enzymes are responsible
for any observed positive response to the assay.
Acetylene: irreversible inhibitor of sMMO
(a)
Methane: competitive reversible inhibitor
(b)
1-pentyne (3.5%): irreversible inhibitor for the 2-monooxygenase pathways
(c)
3-hexyne (2%): irreversible inhibitor for the 3-and
(d)
4-monooxygenases
Phenylacetylene (10-15%): dioxygenase
(e)
DNA…
(f)
Additional Control Assays
To offer supporting evidence for the enzyme activity probes.
PCR characterize the potential of the microbial community
TOD: toluene 2,3-dioxygenase
TOL: xylene monooxygenase
RMO: toluene-3,-4-monooxygenase
PHE: toluene-2, -3, -4-monooxygenase
sMMO: mmoX (f882 & r1403)
Universal: (8F and 907R).
FISH characterize the activity of the microbial community
Eubacteria
Cytophaga-Flavobacterium (most common toluene degrading organisms),
type I and type II methanotrophs
component B of the sMMO
…
Common Myths, dismissed
Early studies (1980-mid-90s) Recent studies have shown that under
determined that: natural conditions:
(a) Cometabolism requires a natural (a) Non-aromatic substrates can induce
substrate induction (methane, activity (naturally occurring
phenol, other aromatic) phenolic compounds e.g. humics);
TCE itself can induce cometabolic
(b) Cometabolic degradation of TCE
activity
results in TCE epoxides and/or
oxygen radicals which inactivate (b) Studies have shown that TCE
the active site of the oxygenase epoxides do not cause significant
decreases in TCE cometabolizing
(c) Growth on non-inducing
abilities or rates
substrates will result in an enzyme
that will not cometabolize (c) Growth on non-inducing substrates
chlorinated solvents (TCE) results in TCE degradation
Heald and Jenkins, 1994; McClay et al., 1995; Leahy et al., 1996; Shingleton et al.,
1998; Ryoo et al., 2000, 2001; Lee et al., 2002; Yeager et al., 2004