K.Karthikeyan

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MICROBIAL ENZYMES IN ENVIRONMENTAL CLEAN UP

• K.Karthikeyan
• Research Scholar
• Junior Research Fellow, UGC NET
• Department of Environmental Sciences
• Bharathiar university
• Coimbatore- 641 046

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Enzymes Key concepts

• Proteins - catalyze biological reactions
• Have quarternary structure
• Can chemically / physically denature and renature
  
• Specific to specific reactions except those
  involved in co-metabolic reactions
• Re-available for catalysis after reaction
• Naming indicates type of reaction and substrate
• Intracellular or extracellular
• Most common product - microbes
• Overall value - industrial enzymes- 2.0 billion
  2009.
• In many household items - that you would never
  think to have a biotechnology component

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Fermentation - process - production of useful
products - mass culture of single - cells End
products / various intermediate products /
metabolites siphoned off - purified -
commercial use
Fermenter / Bioreactor
Stirred tank reactor
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FERMENTORS OF VARYING CAPACITY
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Fermentation facility
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Purification
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Desorbing the desired enzyme
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Applications of Industrial Biotechnology

• Industrial use of biological systems (whole
  cells or enzymes)
• Waste recycling
• Biobleaching
• Textile treatment
• Hydrocarbon degradation
• Food enzymes etc.,

• MICROBIAL PRODUCTS
• Amino Acids
• Vitamins
• Food Additives
• Enzymes
• Recombinant Protein Drugs
• Antibiotics
• Bioplastics
• Fuels
• Bioconversion/Biocatalysis
• Agriculture
• Biosurfactants

• What can be cleaned up using bioremediation?
• Oil spills
• Waste water
• Plastics
• Chemicals (PCBs)
• Toxic Metals

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Industrial Biotechnology

• Application - life sciences - conventional
  manufacturing.
• Uses genetically engineered bacteria, yeasts and
  plants - whole cell systems or enzymes
• In most cases results in
• lower production costs
• less pollution
• resource conservation

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Bioremediation reclaiming - contaminated sites
microbes / similar biological agents. Entails -
removal, degradation, or sequestering of
pollutants / or toxic wastes
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Enzyme Name GE Organism Uses
?-acetolactate bacteria Removes bitter
substances decarboxylase from beer ?
-amylase bacteria Converts starch to simple
sugar Catalase fungi Reduces food
deterioration Chymosin bacteria or fungi Clots
casein to make cheese ?-glucanase bacteria Impr
oves beer filtration Glucose isomerase bacteria
Converts glucose to fructose Glucose
oxidase fungi Reduces food deterioration Lipase
fungi Oil and fat modification Maltogenic
amylase bacteria Slows staling of
breads Pectinesterase fungi Improves fruit
juice clarity Protease bacteria Improves
bread dough structure xylanase (hemicellulase) bac
teria or fungi Enhances rising of bread dough
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LACCASE

• Laccases - family of multicopper oxidases
  (cucumbers) and mammalian plasma protein
  ceruloplasmin.
• Isolated in 1883, Rhus venicifera - Japanese
  lacquer tree.
• Laccases - fungi - lignolytic white-rot fungus
  Trametes versicolor.
• Physiological function - still investigation.
• Although - implicated - degradation of the
  biopolymer lignin.
• Bioremediation - soils and water
  -environmentally friendly processes - pulp and
  paper industry.

Ribbon representation X-Ray crystallographic
Trametes versicolor laccase
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LIPASES

• Lipases
• production of carbohydrate esters of fatty acids.
• food and flavor making.
• catalysis of several unnatural reactions such as
  esterification and transesterification.
• synthesis of fatty esters as cosmetic or
  surfactants.

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• Denim jean cellulose from fungi Trichoderma
  reesei and Aspergillus niger - faded look and
  texture.
• Protease subtilisin - Bacillus subtilis -
  component of Laundry detergent - remove and
  degrade protein stains.
• Enzymes - catalytic machinery of living systems.
• Enzymes - fermentation sugar to ethanol by
  yeasts - reaction similar for beer and wine
  manufacturing.
• Enzymes oxidize ethanol to acetic acid- vinegar
  production.
• More than 75 - industrial enzymes - hydrolases.
• Protein-degrading enzymes - 40 of all enzyme
  sales
• Limited number - known enzymes - commercially
  available - even smaller amount - large
  quantities.

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• Intracellular enzymes - purified from thousands
  of different cell proteins and other components.
• Organism - enzymes - GRAS-status - food processes
  .
• Organism - high amount - desired enzyme -
  reasonable time frame.
• Industrial enzymes - Aspergillus and Trichoderma,
  Streptomyces, fungi imperfecti and Bacillus
  bacteria.
• Enzymes which do not have - desired properties -
  industrial application.
• E.g extreme thermo stability or overproduction
  of the enzyme.
• Protein engineering - used - improve commercially
  available enzyme to be a better industrial
  catalyst.
• Proteinases, lipases, cellulases, a-amylases and
  glucoamylases.

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• Bacterial proteinases - detergent enzymes.
• Genetically engineered to make it more stable -
  hostile environment -several different chemicals
  present.
• Cellulases - detergents - since early 90s.
• Cellulase - an enzyme complex - degrading
  crystalline cellulose to glucose.
• Lipase - in powder and liquid detergents to
  decompose fats.
• Lipase is produced in large scale by Aspergillus
  oryzae .
• Amylases - detergents - remove starch based
  stains.

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• Use of enzymes - textile industry - most rapidly
  growing fields in industrial enzymology.
• Starch - long time - been used - protective glue
  of fibers - weaving of fabrics. This is called
  sizing.
• Enzymes - important aspect of animal feed
  industry.
• Poultry - used in pig feeds and turkey feeds.
• Leather industry - proteolytic and lipolytic
  enzymes in leather processing.
• Enzymes - remove - animal skin, hair, and any
  unwanted parts.
• Xylanases - good example - engineered enzyme -
  Trichoderma. biobleaching.

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• Enzyme Technology
• Enzyme technology - how to use enzymes????
• Simplest way in to a process - where they
  catalyse the desired reaction - gradually
  inactivated during the process.
• Immobilize - reused.
• Immobilization - ultrafiltration membranes in the
  reactor system.

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Thermostable Enzymes

• Thermophilic and thermotolerant microorganisms
  -potential sources of suitable enzymes- more
  thermostable - more resistant than their
  mesophilic counterparts - organic solvents,
  detergents, low and high pH, and other denaturing
  agents.
• Reduces the reaction time - limits the
  probability of microbial contamination.
• cause inactivation of undesired enzymes
  originating - food material during the industrial
  process.

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Thermostable enzymes
I. AMYLASES Removal of starch sizer from
textile . II. CELLULASES most abundant
organic compound in the world Biopulping
Tanning of leathers. Production of fuel alcohol
- corn starch - 3.41 billion gallons in 2008
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Wastewater
Treatment of domestic sewage or industrial
waste Utilizes aeration to oxygenate allowing
aerobic microbes to digest solid waste
Before After
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Oil-eating bacteria to clean up toxins in the
environment
Microorganisms - creosote and chlorinated phenols
contaminated site - Florida
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Environmental contamination by explosives
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Growth of Enterobacter cloacae on TNT
Biodegradation of RDX by R. rhodochrous 11Y
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INSECT SOURCE
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Removal of Textile Finishing Bleach Residues

• Hydrogen peroxide - bleaching textiles usually
  requires several rinsing cycles.
• New enzyme process - only one high temperature
  rinse - remove bleach residues.
• Reduced production costs
• Reduced energy consumption by 14
• Reduced water consumption by 18

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Wood pulp process

• Traditional pulping wood chips - boiled in a
  chemical solution to yield pulp.
• Biopulping (treatment of woodchips with a fungus)
  - enzymes - degrade lignin - break down wood cell
  walls.
• If next step is mechanical treatment, result is
  30-40 reduction in energy inputs.
• If next step is chemical treatment, result is 30
  more lignin being removed and lower amounts of
  chlorine bleach used.
• Cost reduction due to savings on energy and
  chemical costs.

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Wood Pulp Brightening
Phanerochaete rivulosus

• Wood pulp digestion is followed by bleaching in a
  multi-stage process to yield bright, strong pulp.
• Two options to reduce chlorine
• 1) reduce lignin prior to bleaching (enzymes
  still in RD)
• 2) change bleaching chemistry
• Xylanase treatment reduces the use of bleaching
  chemicals by 10-15 and reduces toxic dioxin
  formation.

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Zinc Refining

• In old process - finishing wastewater - heavy
  metals, sulphuric acid and gypsum - precipitate
  sulphates.
• New biological process - sulphate reducing
  bacterial enzymes - sulphate reduction.
• Zinc and sulphate to - zinc sulphide - recycled
  to the refinery.
• Result - no gypsum is produced -water quality -
  improved - valuable zinc is recycled.

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Bioleaching of Copper Ore

• Copper smelters - generally heavy polluters.
• Bacterial enzymes - leaching metals from ores.
• Treat low - grade ores or concentrates.
• Biological leaching -environmental benefits-
  lowers environmental emissions and costs.
• Reduces generation - particulate emissions
  (dust).
• Bacteria - sulphur dioxide emissions.
• Allows - safe handling of arsenic impurities.

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Detergent Enzymes
Detergents - largest application of industrial
enzymes Traditionally - lipolases, proteases
amylases Recent innovation - mannanase Aids in
removing stains containing guar gum Enzymes -
engineered - stability - detergent, alkaline pH,
and cold water
Subtilisin, a protease used in laundry
detergents The recombinant protein was engineered
to remain active in the presence of bleach Bleach
caused the oxidation of one amino acid
(methionine) and the enzyme lost 90 of its
activity By replacing this amino acid with
alanine, the engineered enzyme was no longer
sensitive to oxidation
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Oil/Wastewater Cleanup
Bacteria degrade organic matter in
sewage. Bacteria degrade or detoxify
pollutants such as oil and mercury
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Exxon Valdez

• Microbes - digest hydrocarbons.
• Naturally occurring microbes oil spill to
  clean shore
• lines
• Approach - Exxon Valdez oil spill accident.
• Stimulated -rate of biodegradation - 2 - 5 X

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• Smaller scale cleanup is feasible
• For 3 months - nutrients microbes - sprayed
  -field
• After 11 months the site was deemed clean

ON SITE
Before After
6000yards3 petroleum 4000ppm 95 ppm
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Directed evolution is the most recent tool
utilized in the creation of new and better
enzymes ( other proteins)
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Key Messages

• Why adopt biotechnology? To cut costs and be
  environmentally friendly.
• Companies -- work with government and stay close
  to the regulators.
• Government -- companies still need help
  especially incentives and R D funding.
• promote the diffusion of this green technology
  into many industrial sectors.
• Government can help the private sector prevent
  pollution AND help companies cut costs
  significantly.

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• Industrial Biotechnology - early stages of
  development.
• Innovative applications - increasing and
  spreading rapidly into all areas of
  manufacturing.
• Useful tools - cleaner and more sustainable
  production methods and will continue to do so in
  the future.
• It is in the interest of both business and
  government to foster the diffusion of these
  innovative applications into many sectors of the
  manufacturing economy.

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Ben Franklin

• An ounce of prevention is worth a pound of
  cure.

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If Ben Were Alive Today he might say

• A pound of pollution prevented is cheaper than
  an ounce of pollution controlled!

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The Triple Bottom Line

• Size of triangle indicator of sustainability

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THANK YOU