RESEARCH NEEDS FOR BIOAUGMENTATION - Bioaugmentation for Groundwater Remediation - page 328

12.2.1 Molecular Scale
On a molecular scale, metabolites and metabolic pathways, enzymes, and their activity and
regulation can be stitched together to create a model of an organism's life cycle. From this
model, some predictions of how to improve or change the organism's activity can be made. For
specific reactions, researchers attempt to identify proteins of interest from deoxyribonucleic
acid (DNA) information and subsequently work to characterize the proteins' activities. In the
field of bioremediation, particular attention has focused on the biodegradative enzymes
responsible for contaminant transformation. These are typically classified as dehalogenases
or oxygenases: families of proteins responsible for the degradation of chlorinated compounds.
In the case of anaerobic reductive dechlorination, current understanding of the biochemical
mechanism at the enzyme level is severely behind what is known for other catalytic enzymes,
such as the aerobic hydrolytic dehalogenases (Chan et al., 2010 ) or the oxygenases (Arora et al.,
2009 ; Nebe et al., 2009 ). Many gene sequences of predicted reductive dehalogenases have been
identified, but this has not led to an understanding of how these enzymes break the carbon-
halogen bond. Considerable effort has been directed at characterizing these enzymes (Maillard
et al., 2003 ; Van De Pas et al., 1999 ), but of the over 280 putative reductive dehalogenase genes
known, only 12 have been biochemically characterized or been identified though molecular
biology techniques (Adrian et al., 2007 ; Bisaillon et al., 2010 ; Cheng and He, 2009 ; Grostern and
Edwards, 2009 ; Krajmalnik-Brown et al., 2004 ; Krasotkina et al., 2001 ; Magnuson et al., 1998 ;
Maillard et al., 2003 ; Marzorati et al., 2007 ; Miller et al., 1998 ; Muller et al., 2004 ; Nakamura
et al., 2006 ; Tsukagoshi et al., 2006 ).
Reductive dehalogenases are a challenge to purify from cultures containing dechlorinating
organisms. These strictly anaerobic organisms do not grow to high cell density, and the
reductive dehalogenases are membrane-associated and oxygen-sensitive, so yields of purified
protein are very low or of an inactivated form, and are thus not adequate for many subsequent
biochemical analyses. Despite these limitations, enzyme assays on crude extracts and partially
purified proteins can yield valuable insights into the kinetics (Adrian et al., 2007 ; Grostern et al.,
2009 ) or the activities of individual enzymes (Adrian et al., 2007 ).
An alternative to de novo purification is heterologous expression - i.e., placing the gene for
the enzyme in another “tamed” organism (such as E. coli ) and instructing this organism to
make large amounts of the protein. To date, this approach has not led to expression of a
functional reductive dehalogenase, but eventually this problem will be solved. Once this is
achieved, researchers will be able to synthesize every predicted reductive dehalogenase enzyme,
test substrate ranges and kinetics, specifically identify all steps in the dechlorination reaction,
and optimize the enzymes for new halogenated contaminants by modifying key amino acids
in the active site. Mutant studies with aerobic (hydrolytic) dehalogenases have enabled con-
struction of dehalogenases with increased efficiency (Bosma et al., 2002 ) and led to the
identification of defluorinases (Chan et al., 2010 ).
In the absence of a purified protein product, a variety of both traditional biochemical and
larger-scale transcriptomic/proteomic studies can be used to identify important proteins in
contaminant-degrading organisms, particularly those whose genomes have been sequenced. For
example, transcriptomic and proteomic studies that examine the complete profile of gene or
protein expression at the organismal level have been of some help in identifying substrate
ranges for some of the reductive dehalogenases (Johnson et al., 2008 ; Morris et al., 2007 ; Rahm
and Richardson, 2008 ). Most recently, substrates for dehalogenases have been identified using
non-denaturing polyacrylamide gel electrophoresis to first partially purify enzymes from crude
cell extracts from mixed or pure cultures. Partially-purified enzymes in gel slices are assayed
for dehalogenating activity, and associated proteins identified using Liquid Chromatography
Tandem Mass Spectrometry (LC-MS/MS) (Adrian et al., 2007 ). This approach is being applied