Molecular Mechanisms Of Phthalic Acid Esters Degradation In Gordonia Alkanivorans YC-RL2

There is growing public concern over the toxicity and carcinogenicity of phthalic acid esters?PAEs?.PAEs are ubiquitous in our environment and have been reported even in the remote parts of Arctic circle.Their semi-volatile and hydrophobic properties allow them to be transported over long distances and persist in the environment.In anaerobic and nutrient-poor environments,PAEs can persist for many years.Within the phylum of Actinobacteria,the recent genus of Gordonia is especially important in PAEs degradation.Though microbial PAEs degradation pathways have been elucidated,genomic information of these strains and enzymes is limited.Furthermore,enzymes capable of hydrolyzing high molecular weight PAEs are rarely reported.The genome of a recently isolated Gordonia alkanivorans strain YC-RL2 was sequenced using Biosciences RS II platform and Single Molecular Real-Time?SMRT?technology.The genome was annotated by NCBI Prokaryotic Genome Annotation Pipeline.The generated genome sequence was 4,979,656 bp with an average G+C content of 67.45%.Calculation of Average Nucleotide Identity?ANI?confirmed previous classification that strain YC-RL2 is indeed G.alkanivorans.The sequences were searched against and COG databases;3,132 coding sequences?CDSs?were assigned to COG families and 1,808 CDSs were predicted to be involved in 111 pathways.95 of the KEGG annotated genes were predicted to be involved in the degradation of xenobiotics.A total of 203 and 22 CDSs were annotated as esterase/hydrolase and dioxygenase genes respectively.A total of 53 biosynthetic gene clusters?BGCs?were predicted by antiSMASH?antibiotics&Secondary Metabolite Analysis Shell?bacterial version 4.0.In this study,Car259 and monoethylhexyl phthalate hydrolase?MehpH?genes encoding for carboxylesterase and MehpH respectively were cloned from the genome of G.alkanivorans YC-RL2 and heterogeneously expressed using pET32a?+?system in Escherichia coli.The effect of environmental factors such as pH,temperature,ions,solvents and inhibitors on Car259 were determined using the standard enzyme assay using p-nitrophenyl esters as substrates.Car259 had an approximate molecular weight of 59 kDa and exhibited optimal activity at pH 8.0 and 45?and hydrolyzed short to medium chained p-nitrophenyl esters.The enzyme showed excellent tolerance to divalent metal ions and organic solvents.It showed a preference for higher molecular PAEs especially di?2-ethylhexyl?phthalate?DEHP?.High-pressure liquid chromatography-mass spectrometry?HPLC-MS?of intermediates showed two major intermediates;phthalic acid?PA?and monoethylhexyl phthalate?MEHP?.Docking using Autodock tools and visualization by PyMOL indicated that Ser200-His429-Glu328 constituted the catalytic triad.Car259 was capable of not only hydrolyzing DEHP but also two ester bonds of PAEs.We investigated the effect of environmental factors on a MehpH activity using the standard enzyme assay.MehpH showed a different optimum temperature and pH?40°C and 8.0,respectively?.The enzyme was remarkably stable in presence of organic solvents,detergents and ions.However,it was inhibited by 2 mM of Ni²⁺,Fe³⁺,Cu²⁺,Zn²⁺ions,1 mM phenylmethylsulfonyl fluoride?PMSF?,0.5 mM paraoxon,1 mM phenyl glyoxal?PGO?,2 mM diethyl pyrocarbonate?DEPC?and 5 mM eserine.The pentapeptide motif GXSXG and catalytic triad DSH conserved in serine hydrolases were present in the sequence.The docking result showed that the amino residues Thr152 and Ser230 were much conserved among the hydrolases and closely associated with MEHP?5.8???and 3.6???respectively?and thus may play important roles in the catalytic process.However,MEHP was not very close to the catalytic triad acid residues Ser125,His291,Asp259.The results of these studies suggested that Gordonia as a genus has a great and un-tapped biotechnological potential that could be utilized further.The results also show that Gordonia alkanivorans strain YC-RL2 has a great potential in bioremediation of xenobiotics especially PAEs.