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Tuning biphenyl dioxygenase for extended substrate specificity.
1Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA.
Biotechnology and Bioengineering
|July 9, 1999
Summary
Engineered biphenyl dioxygenase enzymes show enhanced degradation of highly substituted polychlorinated biphenyls (PCBs). This breakthrough offers a promising strategy for bioremediation of persistent PCB pollutants.
Area of Science:
- Environmental Microbiology
- Biotechnology
- Enzyme Engineering
Background:
- Highly substituted polychlorinated biphenyls (PCBs) are persistent organic pollutants resistant to aerobic biodegradation.
- The initial aerobic degradation step is catalyzed by biphenyl dioxygenase, often a rate-limiting step.
- Existing biphenyl dioxygenases exhibit specific substrate preferences, limiting their effectiveness against diverse PCB congeners.
Purpose of the Study:
- To engineer biphenyl dioxygenase variants with an expanded substrate range for enhanced polychlorinated biphenyl (PCB) degradation.
- To investigate the role of specific amino acid substitutions in altering enzyme substrate specificity.
- To develop novel biocatalysts for the bioremediation of recalcitrant PCB mixtures.
Main Methods:
- Functional evolution of the bphA gene using DNA shuffling and staggered extension process (StEP).
- Selection and characterization of engineered biphenyl dioxygenase variants.
- Sequence analysis of successful variants to identify key mutations.
- Assessing degradation capabilities against various PCB congeners and commercial mixtures (Aroclor 1242, Aroclor 1254).
Main Results:
- Several engineered biphenyl dioxygenase variants demonstrated an extended substrate range, degrading both ortho- and para-substituted PCBs effectively.
- Variants showed superior degradation of tetra- and pentachlorinated PCBs and commercial PCB mixtures.
- Sequence analysis revealed 4-6 template switches in most variants, with Thr335Ala and Phe336Ile substitutions being critical for altered substrate specificity.
- The engineered enzymes significantly improved upon the substrate specificity of parental enzymes from Burkholderia cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.
Conclusions:
- DNA shuffling and StEP are effective strategies for evolving biphenyl dioxygenase for improved PCB degradation.
- Specific amino acid substitutions, particularly Thr335Ala and Phe336Ile, are crucial for broadening substrate specificity.
- Engineered dioxygenases hold significant potential for the bioremediation of diverse and highly substituted polychlorinated biphenyls.