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Substitution of individual residues in region III of Bph A, comprising Thr335, Phe336, Asn338, and Ile341, improved the ability of the enzyme to transform 4,4′-dichlorobiphenyls, although the greatest improvements in activity were achieved by multiple substitutions in this region, suggesting that there is a cooperative or additive effect (4).
Several studies have since confirmed the importance of these residues in determining the enzyme's congener preference and regiospecificity (3-5, 36, 54). The steady-state kinetic parameters for biphenyl of anaerobically purified non-His-tagged enzymes were determined, and the activities towards various PCB congeners were investigated.
The microbial degradation of biphenyl has been well studied as a potential means of remediating soils contaminated with polychlorinated biphenyls (PCBs) (46).
DNA polymerase was purchased from Roche (Laval, Quebec, Canada). Acetonitrile, ethyl acetate, and hexane (Fisher Scientific, Mississauga, Ontario, Canada) were high-performance liquid chromatography (HPLC) grade. strains DH5α (27) and C41(DE3) (42) were used for DNA propagation and protein overexpression, respectively.
For example, BPDO-substituted congeners containing up to six chlorines (10, 43).
This enzyme has the relatively unusual ability to catalyze the 3,4-dihydroxylation of certain 2,5-substituted congeners, such as 2,2′,5,5′-tetrachlorobiphenyl (32).
Culture media were inoculated with 1% (vol/vol) of an overnight culture and grown at 37°C until the optical density at 600 nm (OD-galactopyranoside (IPTG) to a final concentration of 1 m M, and each culture was transferred to 20°C (LB400, II9, and II10) or 25°C (B356) for an additional 18 h before it was harvested by centrifugation.
The harvested cell pellet was washed twice with 500 ml of 25 m M HEPES (p H 7.3) containing 10% glycerol and frozen at −80°C until use. Plasmid DNA was purified using a Quantum Prep kit (Bio-Rad, Mississauga, Ontario, Canada).
PCBs are aerobically transformed by the pathway, a pathway comprising four enzymes that initiates the catabolism of biphenyl.