populations can be of immense value in bioremediation of persistent chlorinated
compounds especially the PHAs and PCBs.
Gottschalk and Knackmuss (1993) designed a mixed culture of Pseudomonas
spp. N31 and B13 that oxidized 4-chloro-2-nitro phenol and its toxic metabolite
4-chlorophenol involving oxygenase of Pseudomonas spp. N31 and
4-chlorocatechol dioxygenase of Pseudomonas spp. B-13, respectively. However,
use of bacterial consortia resulted in the formation of undesirable amount of dark
colored toxic metabolites. The presence of chlro- and methyl-arenes at that siteinduced meta- and ortho-pathways caused misrouting of methyl- and chloro-arenes.
This leads to substrate incompatibilities and production of metabolic dead-end
products, which are toxic to the bacterial cells culminating in the cessation of
mineralization activity.
2.9.3 Modification of Substrate Specificity by Manipulations
of Enzymes
Small modifications to a catabolic gene sequence may alter the properties of the
encoded enzymes. The enzyme substrate specificity or gene transfer specificity can
be altered by substitutions of single base pairs or point mutations. Deoxygenases and
dehalogenases are the two enzymes that have been modified by enzyme bioengineering. By comparison of amino acid sequences and models of tertiary structures of
haloalkane dehalogenases, their active centers were identified and selected as possible targets for site directed mutagenesis. Erickson and mondello (1993) reported
that biphenyl deoxygenases of Pseudomonas spp. LB 400 possess broad substrate
specificity, whereas the high efficiency of enzyme was reported in
P. pseudoalcaligenes strain KF707. The enzyme showed 95.6% amino acid
sequence similarity in the large subunit, but had different substrate specificities. A
site-directed mutagenesis of four nucleotides that cause a change in these four amino
acid sequences of biphenyls bphA gene (encoding dioxygenase reductase component) was performed. It resulted in a novel dioxygenase that combined the broad
substrate specificity of Pseudomonas spp. LB 400 and efficiency of homologous
enzyme from P. pseudoalcaligenes KF 707 to degrade a range of di-, tri- and tetrapara-substituted polychlorinated biphenyls. Bosma et al. (2002) reported heterologous expression of haloalkane dehalogenase gene dhaA of Rhodococcus spp. M15-3
by mutating the enzyme at two amino acid levels by Cys 176!Tyr and Tyr
273!Phe (phenylalanine) substitutions. The mutated enzyme was placed under
the control of a constitutive promoter in 2,3-dichloro-1-propanol, employing
Agrobacterium radiobacter AD1. The engineered pathway could completely
degrade TCP (1,2,3-trichlorpropane), a waste product from epichlorhydrin
manufacture.
58
A. Sehrawat et al.
compounds especially the PHAs and PCBs.
Gottschalk and Knackmuss (1993) designed a mixed culture of Pseudomonas
spp. N31 and B13 that oxidized 4-chloro-2-nitro phenol and its toxic metabolite
4-chlorophenol involving oxygenase of Pseudomonas spp. N31 and
4-chlorocatechol dioxygenase of Pseudomonas spp. B-13, respectively. However,
use of bacterial consortia resulted in the formation of undesirable amount of dark
colored toxic metabolites. The presence of chlro- and methyl-arenes at that siteinduced meta- and ortho-pathways caused misrouting of methyl- and chloro-arenes.
This leads to substrate incompatibilities and production of metabolic dead-end
products, which are toxic to the bacterial cells culminating in the cessation of
mineralization activity.
2.9.3 Modification of Substrate Specificity by Manipulations
of Enzymes
Small modifications to a catabolic gene sequence may alter the properties of the
encoded enzymes. The enzyme substrate specificity or gene transfer specificity can
be altered by substitutions of single base pairs or point mutations. Deoxygenases and
dehalogenases are the two enzymes that have been modified by enzyme bioengineering. By comparison of amino acid sequences and models of tertiary structures of
haloalkane dehalogenases, their active centers were identified and selected as possible targets for site directed mutagenesis. Erickson and mondello (1993) reported
that biphenyl deoxygenases of Pseudomonas spp. LB 400 possess broad substrate
specificity, whereas the high efficiency of enzyme was reported in
P. pseudoalcaligenes strain KF707. The enzyme showed 95.6% amino acid
sequence similarity in the large subunit, but had different substrate specificities. A
site-directed mutagenesis of four nucleotides that cause a change in these four amino
acid sequences of biphenyls bphA gene (encoding dioxygenase reductase component) was performed. It resulted in a novel dioxygenase that combined the broad
substrate specificity of Pseudomonas spp. LB 400 and efficiency of homologous
enzyme from P. pseudoalcaligenes KF 707 to degrade a range of di-, tri- and tetrapara-substituted polychlorinated biphenyls. Bosma et al. (2002) reported heterologous expression of haloalkane dehalogenase gene dhaA of Rhodococcus spp. M15-3
by mutating the enzyme at two amino acid levels by Cys 176!Tyr and Tyr
273!Phe (phenylalanine) substitutions. The mutated enzyme was placed under
the control of a constitutive promoter in 2,3-dichloro-1-propanol, employing
Agrobacterium radiobacter AD1. The engineered pathway could completely
degrade TCP (1,2,3-trichlorpropane), a waste product from epichlorhydrin
manufacture.
58
A. Sehrawat et al.