Bacterial degradation of 2.4-dichlorophenol : catabolic genes detection and enzyme characterization.

Masters Degree. University of KwaZulu-Natal, WestvilleThe widespread use of chemicals and their frequent release into the environment is the major cause of pollution, worldwide. Chlorophenolic compounds, such as 2,4-dichlorophenol (2,4- DCP), are classified as priority pollutants due to their recalcitrance, persistence and toxicity. They are also of environmental and human health concern due to their carcinogenic and mutagenic properties. The aims of this study were: to isolate 2,4-DCP degrading microorganisms indigenous to contaminated sites in Durban, South Africa, and establish their degradation potential, to detect the presence of 2,4-DCP catabolic genes in selected isolates, and to characterize the enzymes involved in the degradation process. Following enrichment in mineral salt medium (MSM) supplemented with 2,4-DCP (40 ppm) as the sole carbon and energy source, three 2,4-DCP degrading bacteria were isolated and identified as Pseudomonas chlororaphis strain UFB2, Klebsiella pneumoniae strain KPNIH39 and Klebsiella pneumoniae strain DHQP1002001 based on the 16S rRNA gene sequence analysis. These isolates were able to degrade between 49.01% and 75.11% of 2,4-DCP within 10 days, with the degradation rate constant ranging between 0.07 and 0.14 mg/L/d. The PCR amplification of the catabolic genes involved in 2,4-DCP degradation revealed the presence of the phenol hydroxylase (600 and 715 bp), catechol 1,2-dioxygenase (467 and 507 bp), muconate isomerase (651 and 494 bp), cis-dienelactone hydrolase (491 and 567 bp), and trans-dienelactone hydrolase (491 and 567 bp) in Pseudomonas chlororaphis and Klebsiella pneumoniae respectively. The absence of catechol 2,3-dioxygenase gene in these isolates suggests that the organisms most likely follow ortho-pathway for 2,4-DCP degradation. This was further confirmed by enzyme assays from the crude extracts of the isolates. The extracts showed specific activities (in mU/mg protein) of 21840, 15630, 1480, 2340 and 1490 for phenol hydroxylase, catechol 1,2-dioxygenase, muconate isomerase, cis-dienelactone hydrolase and trans-dienelactone hydrolase, respectively. The crude extracts did not show any catechol 2,3-dioxygenase activity. Phenol v hydroxylase (61.6 kDa) and catechol 1,2-dioxygenase (35 kDa) enzymes were purified from the crude extracts of Pseudomonas chlororaphis UFB2 by anion exchange (1mL HiTrap ANX column) and gel filtration (Sephacryl HR100) chromatography to a purification fold of 1.6 and 1.5, and a yield of 57.69% and 13.02%, respectively. Phenol hydroxylase was observed to be optimum at pH 8 and temperature of 30°C while catechol 1,2-dioxygenase was optimum pH 7.5 and optimum temperature of 30°C. The structural predictions for phenol hydroxylase and catechol 1,2-dioxygenase was conducted by tryptic digestion and mass spectrometry and showed two models for each enzyme. Models for phenol hydroxylase were 2-hydoxybinyl-3- monooxygenase (19.18%) and 3-hydroxybenzoate hydroxylase (19.53%). Models for catechol 1,2-dioxygenase were homogentisate 1,2-dioxygenase (81.06%) and 3 oxalate decarboxylase oxide (14.62%). The biophysical properties of phenol hydroxylase from Pseudomonas chlororaphis UFB2 showed that it is not stable and it is highly hydrophilic in water while catechol 1,2-dioxygenase was stable and highly hydrophilic. This study will therefore assist in contaminated sites and alleviate pollution of chlorophenolic compounds in the environment