case enzyme technology
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biotechTRANSCRIPT
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Enzyme technology: from discovery of enzymes to development of
production strains
Mette Lübeck
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What are enzymes?
Enzymes are:
– biological catalysts in the form of proteins, which drive chemical reactions in living cells
– fully biodegradable, replacing chemicals in industrial production
– reducing resource usage and increasing efficiency
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Enzymes as Industrial Products
• Exoenzymes
– Enzymes that are produced in such large amounts that
they are excreted into the medium instead of being held
within the cell; they are extracellular
– Can digest insoluble polymers such as cellulose, protein,
and starch
• Enzymes are useful as industrial catalysts
– High substrate specificity
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Enzymes as Industrial Products
• Enzymes are produced from fungi and bacteria
– Bacterial proteases are used in laundry detergents (can also
contain amylases, lipases, and reductases)
• Isolated from alkaliphilic bacteria
– Amylases and glucoamylases are applied in 1. generation
bioethanol (starch based ethanol)
• Isolated from filamentous fungi
– Cellulases are applied in 2. generation bioethanol
• Isolated from filamentous fungi
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Industrial Enzymes:
An enzyme that can be produced in yields sufficient to use in large scale industrial processes in such a way as to be relatively pure and economical to produce.
>98% of such enzyme products are secreted enzymes
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Microbial Enzymes and Their Applications
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Enzymes as Industrial Products
• Extremozymes
– Enzymes that function at some environmental extreme
(i.e., pH or temperature)
– Produced by extremophiles
e.g. Acid-Tolerant Enzymes
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Figure 25.15b
Examples of Extremozymes: Thermostable Enzymes
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Strategies
1. Screening for enzyme activities
a) classical
b) metagenomic
2. Other screening (e.g. signal trapping)
3. Targeted gene finding
a) in genomic data
b) in cDNA libraries
4. Synthetic biology
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Enzyme discovery
The first step in enzyme discovery is to determine the operating conditions of the industrial enzyme.
This includes:
Is the enzyme intended to improve the performance of an existing product?
Is the enzyme intended for a new application?
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Enzyme discovery
Important parameters that should be addressed for the enzyme:
Importancy of the stability of the enzyme.
Identification of potential inhibitors that can inhibit the enzyme activity.
The activity and specificity of the enzyme.
Temperature profile in the operating conditions.
pH optimum in the operating conditions.
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Classical screening for enzymes
Examples:
Enzymes for detergent use must show optimal performance at high pH and temperatur of 20-50oC.
Some feed enzymes must show extreme stability at high temp. because they are formulated together with the feed. At the same time they have to resist low pH because of the animal stomach.
For biomass conversion, enzymes are interesting that work at high temperature because they in general show higher stability and higher efficiency. This will reduce the cost of enzyme production.
Mette Lübeck
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Classical Screening
Classical microbial screening is based on culturing a broad diversity of microorganisms – based on a variety in
taxonomy
phylogeny
physiology
biochemistry
ecology
The trick is to make a screening strategy that reflects the desired properties of the enzymes or organisms
Mette Lübeck
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Exotic microorganisms from exotic envirnonments?
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Microbial Biodiversity
• only the tip of the iceberg is known so far
• 1 g soil contains > 4000 different genomes
• so far only 1-5 % of all microbes have been cultured
• 3,5 billion years of evolution
• all ecological habitats are populated with microbes
• adaptations to all environmental challenges
• enormous potential of yet undiscovered phyiological & biochemical traits
• Bacteria
• Fungi
Described Species
– 72.000
– 4.000
Estimated Species
– 1.500.000
– 2-3.000.000
Mette Lübeck
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Environmental sampling
Collection of environmental samples: • Enrichment in the lab • Enrichment in situ • Isolation of microbes • Pure cultivation in the Laboratory
Mette Lübeck
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Culture collections
The DSMZ microbial collections contain over 13 000 cultures representing some 6 900 species and 1 400 genera (Archaea, Bacteria, plasmids, phages, yeasts, fungi). Unique subcollections are held in the prokaryotes groups of acidophiles, alkaliphiles, halophiles, methanogens, myxobacteria, phototrophs, thermophiles, and sulfate reducers. Selected strains for antibiotic testing, quality control, bioassay, bioremediation, and strains with certificate are included.
The ATCC Bacteriology Collection is the most diversified assemblage of prokaryotes in the world, containing nearly 18,000 strains in more than 750 genera. Every important prokaryotic physiological group is represented. The Collection holds more than 3,600 type cultures of validly described species, forming the basis for systematic bacteriology, and nearly 500 bacteriophages.
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Classical screening
Secreted enzymes from microorganisms (fungi, bacteria and archaea) have the highest probability to live up to the often extreme conditions.
In contrast to intracellular enzymes, the secreted enzymes have evolved to work in (often extreme) environments by natural selection over thousands of years.
For a ”typical” microbial genome ,1-3% of the genes encode secreted
proteins
Mette Lübeck
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Screening for enzyme activity
There are several parameters that can be tested for:
Different substrates
Natural substrates
Synthetic substrates
Incubation conditions
Different Temperatures
Different pH
Different methods to test enzyme activity
Mette Lübeck
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Screening for enzyme activity
Growth conditions inducing enzyme production Growth on selective media
Agar plates 96wells 384wells 1536wells
Detection of enzyme activity
High through put
Mette Lübeck
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Screening for enzyme activity
A common way is development of a simple assay: e.g. We are interested in identifying microorganisms that are able to efficiently
degrade the polysaccharides in Sugarcane Bagasse to monosaccharides. Therefore we are interested in identifying e.g. fungi that express high
amounts of cellulases, and xylanases when they grow on Sugarcane Bagasse.
We therefore constructed a medium containing Sugarcane Bagasse as the
only carbon source, and identified a specific set of parameters (fixed temperature, pH, cultivation time) for the screening process.
Under these conditions we screened hundreds of fungi in order to identify
the best enzyme producers.
Mette Lübeck
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Identification of good enzyme producers
During the first screening program, several good enzyme producers were identified.
Next step is to identify more specific parameters for the enzymes of interest. This could be conditions under which the enzymes are intended to function: High temperature (e.g. 50-55oC) Low pH (< 4.5) High pressure (2 bar) Environment with many proteases The second screening setup could therefore include these parameters in order to
identify the best or the 5-10 best enzyme producers.
Mette Lübeck
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Development of better enzyme producers
During the second screening setup a few microorganism are identified, that are able to grow under the conditions selected.
Some are growing well, but do not produce enzymes in high yields.
Some do not grow well, but produce good yields of enzyme compared to the growth rate.
Next step could be to mutate the selected strains to grow better and produce enzymes in higher amounts.
Different possibilities exists in order to make mutations in the genome of a microorganism.
Mette Lübeck
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Genome walking
Isolation of fungi from different habitats
Isolation of genes encoding selected enzymes
Cloning and expression of selected genes in a relevant host
Enzyme activity studies
Evaluation of biomass conversion efficacy of enzymes
Screening of fungi for enzyme activity
Enzyme discovery for biomass conversion
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Production
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Improving strategies
Three different strategies for improving the properties of individual cellulase components:
1) Mutagenesis of cells or spores by UV or chemicals.
2) Mutagenesis of specific genes using ”Rational design” in which detailed knowledge to the protein structure and structure-function relationship is required (lecture in Fungal Biotechnology).
3) Mutagenesis of specific genes using ”Directed evolution” (irrational design) in which no knowledge to the protein structure and structure-function relationship is required (lecture in Fungal Biotechnology).
Mette Lübeck
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Elevate the yield by classical mutagenesis
Selected transformants are mutated by:
• UV light.
• Gamma light.
• Chemical mutagenesis, e.g. nitrosoguanidine.
Mutants is tested for:
• Higher yield.
• Loss of specific enzyme activity.
• Loss of specific metabolites.
• Changed morphology.
Mette Lübeck
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1 pH 12
100oC
5oC
Higher temp. tolerance Higher stability Different pH optimum
Low temp. tolerance Low stability
Evolution by mutagenesis
Mette Lübeck
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Screening for good growth and high yield producing mutants
UV
Mutagenese of spores or cells
growth i microtiter plates
isolation of good mutants
test in shake kolbs test i lab.tanks
assay
test i pilot / production
Mette Lübeck
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Why use recombinant technology to enzyme production
Many enzymes cannot be produced in sufficient amounts by classical methods
Many donor organisms are not ideal for fermentation in steel fermentors
Using a few selected host organisms it is possible to develop a high expertise in handling these in such a way that they will be ideal for production
The host strain can be modified in such a way that only the desired product is produced.
Mette Lübeck
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Classical Screening
Advantages:
The biodiversity is enormeous in nature - new organisms and enzymes can be found.
Culture collections contain many organisms – better enzymes can be found.
Gene banks contain many genes – information in order to develop variants – e.g. better enzymes.
Disadvantages:
Time consuming process – easy to
overlook superior organisms ”needle in a haystack”.
It is expensive to keep a culture collection.
Large collection of genes – often lack of heterologous systems for gene expression.
Mette Lübeck
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Fungal genome sequencing projects
www.cazy.org
Mycoparasites Cellulolytic fungi Plant pathogens/ molds Mycorrhiza White rot fungi Brown rot fungi Yeasts Truffle (eatable)
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Fungal genome sequencing projects BG CBHs EGs
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The uncultivated world
Molecular, culture-independent methods has revealed a microbial diversity in soil and sea water that was unknown 2 decades ago.
New divisions of bacteria and Archaea has been discovered and it is thought that only less than 1 % of prokaryotes in soil are cultivable.
Also new fungi has been discovered.
Molecular biology methods provides powerful new approaches to study the microbial diversity in the biosphere.
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Metagenomics
Metagenomics is the genomic analysis of the collective genomes of an assemblage of organisms (enviromental genomics, community genomics).
Metagenomic libraries contains DNA extracted directly from the environment providing sequences, giving phylogenetic and functional information.
Phylogenetic markers (e.g. 16S rRNA for bacteria, 18S or ITS rRNA for fungi) provide indications of the origin of the DNA.
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Microbial Species Diversity
Figure 23.1
High Species Richness and Low to Moderate Abundance
Low Species Richness and High Abundance
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Classical versus metagenomic screening
Lorenz et al., 2002.
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Cloning of genes (lecture in Fungal Biotechnology )
Grow culture
Isolate DNA Restriction enzyme digest Ligate into
plasmid vector
Transformation of host bacterium
Transformation of fungal host
Mette Lübeck
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Proteins
Protein P 1
Protein P 2
Signal Sequence Secreted
Not secreted
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Expression vectors
• A selection marker in order to select Aspergillus transformants is necessary
• amdS: acetamid to acetic acid and ammonium • Function as selection marker if no other ammonium is present.
•A strong promoter is necessary. •Amylase hybrid promoters are very strong artificial
promoters.
• A terminator is necessary in order to terminate gene transcription
• Tamg is a strong terminator
Expression vector: • Well characterised • Minimal in size • Allow efficient expression
plasmid 7643 bp
E. coli selection
Gene of interest
amdS
Promoter
ColE1 origin
Tamg
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Optimisation of protein expression
Protein P 1
Protein P 2
Protein P 3
host 1 host 2 host 3 host 4
Different promoters / Different signal sequences / host combinations Evaluation of yield
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Screening for growth and high yield producing transformants
growth i microtiter plates
isolation of suitable transformants
test in shaking flasks
test i lab tanks
assay
test in pilot / production
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SDS-PAGE of transformants grown in shake flasks
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Strain characterization in lab fermentors
• Tank size 2 – 10 L
• Computer control and data
• Model experiments in well defined medium
• Downscaling of production fermentation methods
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Fermentation proces
• Use of GM microorganisms
• GM microorganisms are used as cell factories for production of enzymes
• GMOs (production strains) are only helpers in the process
• The product is separated
from the GM microorganism
in order to make a clean product
without GMO.
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Diagram for enzyme production
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Harvest and test extracellular enzymes
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1.5 ml
10,000 rpm; 5 min
1 ml
15 µl
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AZurine-CrossLinked Polysaccharides - how does it work?
Remazolbrilliant Blue R
AZCL substrates for enzyme assays
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AZCL-Cellulose – an example
1. Aspergillus – CF - agar
2. Fusarium – CF - agar
3. Penicillium – CF - agar
4. Trichoderma – CF - agar
5. Aspergillus – SB - agar
6. Fusarium – SB - agar
7. Penicillium – SB - agar
8. Trichoderma – SB - agar
9. Aspergillus – SBP - agar
10. Fusarium – SBP - agar
11. Penicillium – SBP - agar
12. Trichoderma – SBP - agar
13. Aspergillus – CF - liq
14. Fusarium – CF - liq
15. Penicillium – CF - liq
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Mette Lübeck 50
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Comparison of different enzyme profiles
arabinan
cellulose xylan
galactan
Mette Lübeck 51
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T. asperellum Enzyme production on different substrates
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Results from transcriptome analysis
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Comparison between the transcriptome based secretome and wheat bran composition
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Conclusions from the paper
• Wheat bran was the best medium for enzyme production
• In total 175 glycosyl hydrolases from 48 GH families were produced