Bio Catalysts

Bio-catalysts
in Industrial and Environmental
biotechnology
– microorganisms
– enzymes
Selection and improvement

Giovanni Sannia
Università degli Studi di Napoli Federico II
Dipartimento di Chimica Organica e Biochimica

Microorganisms
• Where are they found?
• How diverse are they?
• Role in geochemical nutrient cycles.
• How do they grow and what are their requirements for
growth and biodegradation?

Microorganisms in waste treatment:
• Biodegradation and environmental clean up.
• Microbial production and products in industry

Giovanni Sannia

Microorganisms in the environment;
Challenging conventional views of life.
• Sagan and Margulis (1998) “Garden of Microbial Delights”.
– “ALL of the elements crucial to global life- oxygen,
nitrogen,phosphorus, sulfur, carbon- return to a usable
form through the intervention of microbes… Ecology is
based on the restorative decomposition of microbes and
molds, acting on plants and animals after they have died
to return their valuable chemical nutrients to the total
living system of life on earth”
• Gould (1996) “Life’s Grandeur” The Power of the Modal
Bacter.
– The first multicellular organisms do not enter the fossil
record until about 580 million years ago - this is after
about five sixths of life’s histroy have passed. Bacteria
have been the stayers and keepers of life’s history.

Giovanni Sannia

Microorganisms in the environment;
Challenging conventional views of life.

•Sagan and Margulis (1998) “Garden of Microbial Delights”.
–“ALL of the elements crucial to global life- oxygen,
nitrogen,phosphorus, sulfur, carbon- return to a usable
form through the intervention of microbes… Ecology is
based on the restorative decomposition of microbes and
molds, acting on plants and animals after they have died
to return their valuable chemical nutrients to the total
living system of life on earth”
•Gould (1996) “Life’s Grandeur” The Power of the Modal
Bacter.
–The first multicellular organisms do not enter the fossil
record until about 580 million years ago - this is after
about five sixths of life’s histroy have passed. Bacteria
have been the stayers and keepers of life’s history.

Giovanni Sannia

Microorganisms

Where are they found?
Diverse environments
• Virtually every environmental niche
• Extremes of pH and salinity
• Extremes of temperature and pressure
• Without air (Anaerobic)
• Growth on many chemical substrates
• Attached to surfaces in biofilms
• Geothermal vents and subterranean deposits

Giovanni Sannia

Microorganisms

Where are they found?
Biomass on the planet.
• Most culturing analysis misses over 99% of the
microbial population.
• Molecular techniques now reveal hidden diversity
• Heterotrophs - 5-20% biomass in sea waters - up
to 80% of the primary production
• Rich bacterial communities in sub-surface strata
(600 m deep) - up to 2 x 104 tons - more than all
flora and fauna -equivalent to 2 m layer over
planet!

Giovanni Sannia

Microorganisms

How diverse are they?
Plants &
Eubacteria Animals Archaea

• Diverse range of species
• Earliest life on the planet
• Anaerobic then aerobic
• Three Kingdoms
• Eukaryote Plants & Animals
• Eubacteria
• Archaebacteria
• Exteme living bacteria

3 billion years

Giovanni Sannia

Microorganisms
How diverse are they?

Diversity of bacteria in soil

16s rRNA sequences reveal true
diversity in soil DNA
Giovanni Sannia

Microorganisms

Role in geochemical
nutrient cycles.
• Microorganisms play a role as:
•PRIMARY PRODUCERS
•BIODEGRADERS AND CONSUMERS
• Critical role in cycles of many elements;
• Carbon and and Oxygen cycle
• Nitrogen cycle
• Sulfur cycle
• Phosphorus cycle

Giovanni Sannia

Microorganisms

How do they grow: requirements
for biodegradation?

• Nutrients
• Carbon, Nitrogen, Phosphorus, Sulfur
• Many chemicals supply these
• Micronutrients/ trace metals/ vitamins
• Electron acceptors - usually O2
• Converts / burns carbon substrate to CO2
Energy and biomass ie GROWTH

Giovanni Sannia

Microorganisms

Biodegradation
O2 consumption GROWTH - CELL
DIVISION
2.0µm
INCREASE IN
BIOMASS
ORGANIC
POLLUTANT
AND NUTRIENTS
(C,P,N,O,Fe,S……) SINGLE CO2
BACTERIUM evolved
Controlled release of energy
Slow Burning!

Giovanni Sannia

Microorganisms
Oxygen and Electron Acceptors: crucial for
Biodegradation reactions in the
environment.
2H+
H2 O
O2
SUBSTRATE

ADP
METABOLISM Pi
ATP H2/2e-

CARBON ENERGY
GROWTH/Biomass
CO2
Giovanni Sannia

Microorganisms

Role of electron acceptors;
rate of biodegradation

O2 NO3- SO42- Fe3+

H2O NO2- H2S Fe2+
N2
0.814V -0.214V -0.185V
0.741V
FAST SLOW
GROWTH GROWTH
Giovanni Sannia

Microorganisms

Anaerobic growth and
biodegradation

Fermented
Organic matter Acetic Acid

+

H2 , CO2
Methanogenesis

CH4 , CO2 , H2O

Giovanni Sannia

Microorganisms

Fixation of oxygen as a first
step in biodegradation
Cell membrane

ReductaseNAP FerredoxinNAP ISPNAP
NAD+
(OX) (OX) (OX)

O2
OH
OH
NADH ReductaseNAP FerredoxinNAP ISPNAP
+ H+ (RED) (RED) (RED)

Cell Biomass
Further degradation
CO2 Giovanni Sannia

Microorganisms
Biological waste treatment;
Managing microorganisms for
environmental cleanup
• 10 x 106 Chemicals
– 8 x 106 Xenobiotic
– 1 x 106 Recalcitrant
• 0.4 x 106 traded at over 50 tonnes per year
• Toxicological/ biodegradative data on only
around 5000-6000

•Municipal waste-water treatment
•Biodegradation of industrial wastes
• petrochemicals, bulk chemical processes
• textiles, leathers
• metals
• Remediation of contaminated land in situ
Giovanni Sannia

Microorganisms

Biological waste-water treatment:
The activated sludge process.

Giovanni Sannia

Microorganisms
Biological waste treatment;
Advanced industrial membrane reactor.
E F F LU E N T F R E E O F P O LLU TA N T

W A S T E - W A T E R C O N T A IN IN G P O L L U T A N T S
Giovanni Sannia

Microorganisms

Cultivation of microorganisms
for industrial use.

Giovanni Sannia

Microorganisms

Cultivation of microorganisms
for industrial use.

Advanced laboratory
fermenters

Giovanni Sannia

Products from Microorganisms:
Overview of range of examples.
• Various foods and drinks
• Enzymes for varied uses (GM enzymes);
biocatalysts
• Engineered proteins ( antibodies )
• Vaccines and antibiotics (secondary metabolites)
• Primary metabolites and bulk chemicals (amino
acids (glutamic acid) and organic acids (acetic
acid)
• Pharmaceuticals and novel chiral chemicals
• Recovery of metals in bioleaching
• Biosensors (use of enzymes to specifically
detect chemicals in medical and )

Giovanni Sannia

Enzymes

Protein engineering

Tailor-made biocatalysts

• The efficient application of biocatalysts requires
the availability of suitable enzymes with high
activity and stability under process conditions,
desired substrate selectivity and high
enantioselectivity

• Rational (re)design versus directed evolution

Giovanni Sannia

Enzymes

Protein engineering

Genetic manipulation techniques

• Large-scale supply of enzymes at reasonable
price

• Identification of new biocatalysts (screening)
doesnot always yield suitable enzymes for a
given synthetic problem

• Computer-aided site-directed mutagenesis
• Directed (molecular) evolution

Giovanni Sannia

Enzymes

Protein engineering

Site-directed mutagenesis

• Requires structural information and knowledge
about relationship between sequence, structure,
function and mechanism
• Very information-intensive
• Rapid progress in NMR / X-ray methods
• Genome sequence information
• Molecular modeling, bioinformatics
• Prediction of selectivity, activity, stability etc.

Giovanni Sannia

Enzymes

Protein engineering

Rational redesign strategy

• Protein structure
• Planning of mutants, SDM
• Vectors containing mutated genes
• Transformation in E. coli
• Protein expression and purification
• Mutant enzyme analysis
• Negative mutants
• Improved mutant enzymes
Giovanni Sannia

Enzymes

Protein engineering

Rational redesign

• Amino acid substitutions often selected by
sequence comparison with homologous
sequences
• Results have to be carefully interpreted
• Minor changes by a single point mutation may
cause significant structural disturbance
• Comparison of 3D-structure of mutant and wild-
type enzyme necessary

Giovanni Sannia

Enzymes

Protein engineering

Directed evolution

• Evolutive biotechnology, molecular evolution

• Random mutagenesis of the gene encoding the
biocatalyst (e.g. by error-prone PCR)

• DNA shuffling: recombination of gene fragments
(staggered extension process or random priming
recombination)

Giovanni Sannia

Enzymes

Protein engineering

Directed evolution strategy

• Random mutagenesis
• Library of mutated genes
• Transformation in E. coli
• Mutant library > 10.000 clones
• Protein expression in microtiter plates
• Selection parameters
• Mutant enzyme and product analysis
• In vitro-recombination, transformation etc.

Giovanni Sannia

Enzymes

Protein engineering

Selection parameters

• Substrate range
• Stability in organic solvent
• Stability towards reaction conditions
• Thermal stability

• High-throughput product analysis

• Robot technology

Giovanni Sannia

Enzymes

Protein engineering

Selection parameters

• Hydrolysis of esters: agar-plate assay based on
pH indicators
• Parallel assaying of replica-plated colonies with
substrate analog
• Isotopically labeled substrates
• Capillary electrophoresis (7000 samples per day)

• Optimization with saturation mutagenesis
Giovanni Sannia

Enzymes

Protein engineering

Improving thermostability

• Cold-adapted proteases
• Combined screening for activity, thermostability,
organic solvent tolerance and pH-profile

• Engineering of entire metabolic pathways
• Phytoene desaturase and lycopene cyclase
shuffling for carotenoid biosynthesis
• Molecular breeding
Giovanni Sannia

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