Pushpa M.Sc biotech.

f Roll no 25

Xenobiotics
Xeno means foreign Biotis means biological These are man made or chemically

synthesized, and natural compound found in unusual amount are xenobiotic compound.

Source of xenobiotic compounds

1. Plastic industry :

2. Pesticide industry : chlorinated and often heterocyclic 3. Paint industry : major ingredient are solvents, xylene, toluene, methyl ethyl ketone, methyl isobutyl ketone and preservatives 4. Others : Electronic industry, Textile industry, Pulp and Paper industry, Cosmetics and Pharmaceutical industry, Wood preservation 5. Petrochemical industry : vinyl chloride and benzene

Xenobiotics may be divided into three categories

Weak xenobiotics Recalcitrant xenobiotics Persistant xenobiotics .

Factor influencing the biodegradation of xenobiotic compounds

Nature of compound. Type no and position of substituents like halogen. Bioavailability to microbes. Concentration of xenobiotic compound. Type and no of other microbes present at site of degradation. Other physiological properties like pH, oxygen concentration, availability of nutrients.

Biodegradation
It means to degrade a complex organic molecule with

the help of biological system. A large no microbes, bacteria, fungal genera posses capability to degrade xenobiotic compound. Pseudomonas are the most predominant group of soil microbes that degrade xenobiotics. Bacteria, they enzymatically convert xenobiotic non halogenated aromatic compound and halogenated compound to either catechol or procatechaute. Than through oxidative cleavage processed to yield either acetyl coA and succinate or pyruvate and acetaldehyde that are readily metabolized by all microorganisms.

Disadvantage of biodegradation
Slow process
Narrow range of degradation Low tolerance

Unavailability of compound to microorganism due to

1.Lack of permeases 2.Strong binding with soil particals 3.Unsoluble in water Contaminated site contain a mixture of compound.

Modification of microbes
1. Enrichment culture technique. 2.Genetic engineering of biodegrdative pathways 1. Manipulate host cell

To enhance the uptake of pollutants Increase the rate of degradation To broaden the range of substrate
Manipulate specific catabolic pathways Total noval or hybrid pathway can be introduced Incomplete pathway can be completed

2.

Strategy used for G.E of microorganisms

Directed mutagenesis Rational design

1. Manipulation by transfer of plasmids

2.
Manipulation by gene alteration

Manipulation by transfer of plasmids

A.K. Chakrabarty and et al in 1970s were create

superbug. 1st patent ever granted for genetically engineered microorganism. This strain is created by transfer of four plasmid, CAM, OCT, XYL and NAH plasmid. Superbug or pseudomonas putida can degrade camphor, octane, xylene and naphthalene.

Cont.
Most of the degradative bacteria that are genetically

manipulated are mesophile but river, lakes and ocean that are polluted generally have temperature range 0 to 20C
To overcome this problem, a TOL plasmid is transfer to

a facultative psychrophile was able to degrade salicylate or having SAL plasmid.

Manipulation by gene alteration

Gene that can be manipulated: Catbolic gene Transport gene Reulatory gene

Degradation of 4-ethylbenzoate
Bringing together different intact plasmid based

degradative pathways to create noval properties. Toluene and xylene degradative pathway of plasmid pWWO. Metacleavage pathway involving xyl operon enable pseudomonas to utilize alkylbenzoate as carbon source. xyl operon have 12 genes. 11 gene are under the control of single pm promoter. pm promoter is positively regulated by xyls gene product which is activated by substrate such as benzoate and 3-methyl bezoate.

Contd.
Bacteria carrying pWWO plasmid could slowly

degrade 4-ethylbenzoate to 4-ethylcatechol, which accumulate and inactivate catechol 2,3-dioxygenase and pathway block. 4-ethylbenzoate does not activate xyls protein.

limitation
1. How to overcome the inactivation of 2,3-

dioxygenase. 2. how to induce transcription of genes of this pathway with 4-ethylbenzoate as the inducer.

Mutant selection for xyls gene

Tetracycline resistant gene is placed under the control

of pm promoter of pWWO plasmid carrying ampicillin resistant gene. The xyls gene was cloned onto another plasmid pNM185 carrying kanamycin resistant gene. Transformants E. coli selected on basis of resistance to both ampicillin and kanamycin. E.coli treated with mutagen ethyl methanesulphonate and grow on a medium containing tetracycline and 4ethylbenzoate. The cells carried altered Xyls protein and interact with 4-etylbenzoate.

Selection of mutated 2,3dioxygenase

Mutated xyls gene subcloned

onto a broad-host range plasmid carrying kanamycin resistant gene and introduced into P. putida cells carrying pWWO plasmid. Cells mutagenized with ethyl methanesulphonate and grow on a medium containing 4ethylbenzoate and kanamycin. Cells grow on this medium have altered catechol 2,3 dioxygenase that was not inhibited by 4ethylbenzoate.

Trichloroethylene degradation
Trichloroethylene used as solvent and degreasing

agent. It is carcinogenic and degraded into vinyl chloride, which is more toxic. Its degradation not require meta cleavage pathway. Toluene dioxygenase is required. Four genes are involved in the toluene dioxygenase synthesis, genes are under the control of tac promoter.

Refrences
Bernard R. Glick, J. Pasternak-principle and

application of recombinant technology. www.wikipedia .com www. Jetking info.com Ncbi.co.in Primrose and tyman, Introduction to Recombinant DNA technology.