The document discusses the use of enzymes in bioremediation. It outlines that enzymatic bioremediation uses isolated enzymes to transform contaminants into less toxic compounds. Extracellular enzymes from white rot fungi have been shown to effectively degrade pollutants like PAHs, PCBs, and dyes. Major enzymes used include lignin peroxidase, manganese peroxidase, and laccase. Case studies demonstrate how these enzymes can decolorize over 90% of textile dyes. While enzymatic bioremediation provides advantages over chemical and microbial methods, further research is needed to reduce costs and improve enzyme stability and activity under various conditions.

1. USE OF ENZYMES IN BIOREMEDIATION
BACHELOR OF TECHNOLOGY (BIOTECHNOLOGY)
BY
SNEHAL.S.MENON
1
DEPARTMENT OF BIOTECHNOLOGY,
STES’S SINHGAD COLLEGE OF ENGINEERING,
VADGAON (BK), OFF SINHGAD ROAD,
PUNE- 411041

2. OUTLINE
Introduction
 Bioremediation
 Enymatic bioremediation
 Enzymological background
 Extracellular enzymes used in bioremediation
 Advantages and disadvantages of extracellular enzymes
 Soluble and immobilized enzymes
 Plants and their associated enzymes in bioremediation
 Major enzymes used in bioremediation
 Case studies
 Scope of enzymatic bioremediation and future prospects
 Conclusion
 References

2

3. INTRODUCTION
ENVIRONMENTAL POLLUTION
Atmosphere
Space
Environment
Earth
Water
3

4. TYPES OF POLLUTION
 Atmospheric
pollution:
pollution of air.
 Water
pollution:
pollution of hydrosphere or water.
 Industrial
effluents pollution:
pollution due to disposal of waste water.
 Domestic
effluent pollution:
pollution due to indiscriminate dispersal of domestic sewage.
 Soil
pollution:
pollution of lithosphere or land.
4

5. OTHER TYPES OF POLLUTION

Noise pollution

Vibration

Noxious odours
5

6. BIOREMEDIATION:


According to E.K. Nyer, the term “bioremediation” refers to all biochemical
reactions of natural attenuation, which includes all biotic and abiotic
processes used to reduce contaminant levels.
Biodegradation is the primary mechanism to reduce biodegradable
contaminants by employing organisms like bacteria, fungi, algae or plants.
Figure 1: The process of waste bioremediation
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.

7. TYPES OF BIOREMEDIATION
 Microbial



bioremediation:
Bioremediation can occur either naturally, or
by the use of bioaugmentation (whole cell introduction) or
Biostimulation approaches (use of nutrients or conditions to stimulate the
native microbial community).
 Enzymatic
bioremediation:
Isolated enzymes may also be used to transform the contaminant into lesstoxic or non-toxic compounds.

Extracellular enzymes:
Extracellular enzymes are either secreted from organisms such as white rot
fungi or are produced during a fermentation process.

Phytoremediation:
It is the in situ use of plants, their enzymatic system, their roots and
associated microorganisms to degrade harmless pollutants present in different
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environmental systems (soil, sediments, groundwater and air).

8. ENZYMATIC BIOREMEDIATION:
 Enzymological background:


Enzymes are biological catalysts that facilitate the conversion of substrates
into products by providing favourable conditions that lower the activation
energy of the reaction.
The regions of the enzyme that are directly involved in the catalytic process
are called the active sites.
8
Figure 2: Mechanism of enzymes

9. EXTRACELLULAR ENZYMES IN BIOREMEDIATION:



Extracellular enzymes refer to those enzymes that are either secreted by the
microbes, such as white rot fungi or those that enter the aqueous phase
during an aerobic submerged fermentation process.
Such enzymes are naturally produced by the microbes and then harvested.
Enzymes from white rot fungi have been shown to be effective
degraders of TNT, phenols, PCBs, PAHs and dyes.
Chrysene(PAHs)
Lindane(pesticide)
Polychlorinated
biphenyls(PCBs)
Figure 3: Some xenobiotics amenable to enzymatic bioremediation
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10. ADVANTAGES AND DISADVANTAGES OF EXTRACELLULAR
ENZYMES
ADVANTAGES
DISADVANTAGES
• Can work in multiple
environments.
• Can be recovered and
recycled.
• Can be used with
different substrates.
• They are biodegradable.
• Difficult
to
maintain
enzyme concentration.
• Difficult to optimize.
• Limits overall success.
• High cost.
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11. SOLUBLE AND IMMOBILIZED ENZYMES

Soluble enzymes

Mobile enzymes can be added at a single point and then spread due to
diffusion, dispersion. and the flows of groundwater and surface water .

Immobilized enzymes

Enzymes can he immobilized onto
granular, fibrous, a tube or a membrane.
a
carrier,
which
can
be
Figure 4: Types of immobilization of enzymes

In general, immobilization makes the enzyme more resistant to
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temperature, pH and substrate concentration swings, giving it a longer
lifetime and higher productivity per active unit.

12. PLANTS AND THEIR ASSOCIATED ENZYMES IN BIOREMEDIATION


The involvement of plants in the bioremediation of pollutants is called as
phytoremediation.
The process of phytoremediation is an emerging green technology that
facilitates the removal or degradation of the toxic chemicals in soils,
sediments, groundwater, surface water and air.
Figure 5: Enzymatic and microbial activities responsible
for enhanced remediation in rhizospheric zone
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13. MAJOR ENZYMES USED IN BIOREMEDIATION

Enzymes from white rot fungi have been found to be very capable of
degrading a large number of different contaminants.

White rot fungi are unique among eukaryotes because they are able to
cleave the carbon-carbon bonds in contaminants such as PAHs.

During the secondary metabolism of plant life, white rot fungi produce and
secrete LiP, manganese peroxidase (MnP) and laccase.

Each of the enzymes can catalyze the one-electron oxidation of phenols and
non-phenolitic substrates.
This results in the production of cation-radical intermediates, which can be
used to futher oxidize non-phenolitic substrates.

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14. 
Lignin peroxidase(LiP)

LiPs are hemoproteins which catalyze reactions in the presence of hydrogen
peroxide.
LiP is very effective in the bioremediation of PAHs.
LiP from Phanerochete chrysosporium. for example. is able to degrade
PAHs.
LiP is also capable of degrading benzo[a]pyrene into 52% 1,6-quinone, 25%
3,6-quinone. and 23% 6,12-quinone.
These product ratios are very similar to those found from the degradation of
benzo[a]pyrene using chemical and electrochemical means.




Benzo[a]pyrene(PAHs)
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15. 
Manganese peroxidase (MnP):

MnP is also a hydrogen peroxide dependent enzyme,but it can only oxidize
organics when in the presence of Mn(Il).
MnP oxidizes Mn(II) to Mn(III), which acts as an obligatory oxidation
intermediate for the oxidation of various compounds.
The Mn(IlI) ions migrate away from the enzyme and start the oxidation of the
lignin and other compounds.


15
Figure 6: Mechanism of MnP

16. 
Laccase:

Laccases are multi-copper oxidases that catalyze the one electron oxidation
of substituted phenols, anilines, and aromatic thiols to the corresponding
radicals with the concomitant reduction of molecular oxygen to water.
These radicals produce polymeric products by self-coupling or crosscoupling with other molecules, and dechlorination, demethoxylation and
decarboxylation during coupling and polymerization of differently
substituted substrates may also occur.
These enzymes appear suitable and versatile catalysts, very useful for the
application in several biotechnological processes.


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Figure 7: Designer laccases

17. OTHER EXTRACELLULAR ENZYMES USED IN BIOREMEDIATION



HRP is a peroxidase that is secreted by the root hairs of the horseradish
plant and can catalyze the oxidation of compounds such as phenols,
biphenols, anilines and benzidines over a large range of pHs and
temperatures .
Extracellular enzymes such as proteases, amylases and lipases are produced
during the aerobic fermentation of organic matter by yeast or other
microbes.
Environments such as waste-water require a different type of enzyme and
these enzymes, rather than catalyzing the oxidation of recalcitrant
compounds, catalyze the degradation of organic matter .
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18. CASE STUDIES
BIOREMEDIATION OF TEXTILE EFFLUENT USING ENZYMES






Enzymes can act on specific recalcitrant pollutants to remove them by
precipitation or transformation to other products.
White-rot fungi were able to degrade dyes using lignin peroxidase (LiP)
and manganese dependent peroxidase (MnP)
The manganese peroxidase produced by Phanerochaete sordida showed
higher range of 90% decolorization of azo and anthraquinone dye.
The comparison of the fungal isolates and enzymatic treatment in the
degradation of reactive blue 5 dyes was carried out and the degrading
capacity of the enzyme manganese peroxidase was 1.5 times greater than
the fungal isolates.
Lignin peroxidase obtained from Phanerochaete chrysosporium is effective
against methylene blue and azure B dyes
VP (Versatile peroxidase) has been recently described as new family of
ligninolytic peroxidases,together with lignin peroxidase and manganese
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peroxidase obtained from Phanerochaete chrysosporium .

19. 





These enzymes exhibited both lignolytic peroxidase and manganese
peroxidase activity and therefore these enzymes were called as hybrid
manganese peroxidase-lignin peroxidase or versatile peroxidase.
Recalcitrant dyes could be successfully decolorized by peroxidases in the
presence of some suitable redox mediators.
Treatment of recalcitrant pollutants by using enzyme-redox mediator system
will be significantly useful procedure for targeting number of dyes with
diversified structures.
Laccase produced by Pycnoporus snnguineus in liquid cultures can
completely decolor bromophenol blue and malachite green (both
triphenylmethane dyes) and partially decolor orange G and amaranth (both
azo dyes).
lmmobilization of the enzyme on alumina increased its thermal stability and
made it less affected by inhibitors, such as halides and dye additives.
lmmobilized laccase was also able to decrease the toxicity of the dyes by up
to 80%
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20. 


The enzymatic decolorization of industrial dyes is a big challenge due to
large diversity of chemical structures.
Enzymes offered several advantages such as greater specificity, better
standardization, easy handle and store and no dependence on bacterial growth
rates.
A major obstacle that will have to he overcome is the long contact time
required for decolorization to occur.
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21. SCOPE OF ENZYMATIC BIOREMEDIATION AND
FUTURE PROSPECTS


The scope of bioremediation is to decrease the concentration of organic
pollutants at undetectable levels or, if measurable, lower than the limits
established as safe or tolerable by regulatory agencies.
There are several fields in which enzymes can be applied:
Figure 8: Overview of enzymology of biological remediation

Enzymatic bioremediation improved with molecular tools can be
particularly suitable for situations where rapid remediation is required.
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22. CONCLUSION






Enzymes present environmental advantages against chemicals and
microorganisms. They are:the biotransformation does not generate toxic side products as is often the
case with chemical and some microbiological processes;
the enzymes are digested, in situ, by the indigenous microorganisms after the
treatment;
the requirement to enhance bio-availability by the introduction of organic
co-solvents or surfactants is much more feasible from an enzymatic point of
view than using whole cells;
the production of enzymes at a higher scale, with enhanced stability and/or
activity and at a lower cost is feasible by using recombinant-DNA
technology .
However, a more extensive effort is required to overcome several
bottlenecks: high enzyme cost, low activity and/or stability under
given conditions, low reaction yields and the low biodiversity in
organisms screened so far.
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23. REFERENCES







Khopkar.S.M, Environmental Pollution Monitoring and Control, Newage
publishers,2007.
Miguel Alcalde, Manuel Ferrer, Francisco J. Plou and Antonio Ballesteros;
2006; Environmental biocatalysis: frommremediation with enzymes to novel
green processes; TRENDS in Biotechnology; Vol.24; No.6; 1-7.
Timothy P. Ruggaber and Jeffrey W. Talley; 2006, Enhancing Bioremediation
with Enzymatic Processes; 1-13.
M.A. Rao, R. Scelza, R. Scotti and L. Gianfreda; 2010; Role of enzymes in the
remediation of polluted environments; Soil science. Plant nutrition, 10(3): 333353
R. S. Peixoto, A. B. Vermelho, and A. S. Rosado; 2011; Petroleum-Degrading
Enzymes: Bioremediation and New Prospects; Volume 2011; 1-7.
Chandrakant S. Karigar and Shwetha S. Rao, 2011, Role of Microbial Enzymes
in the Bioremediation of Pollutants,Enzyme Research, Volume 2011,1-11.
Palanivelan Ramachandran,Rajakumar Sundharam,Jayanthi Palaniyappan and
Ayyasamy Pudukkadu Munusamy, 2013, Potential process implicated in
bioremediation of textile effluents, Advances in Applied Science Research,
4(1),131-145.
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