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Enzyme Discovery and
Engineering
CHEM-E3140 Bioprocess Technology II
30.10.2015
Ossi Turunen
Why to find new enzymes for biotechnology?
Existing enzymes may not be good enough in
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performance
stability
activity in solvents
pH properties
substrate specificity
enantioselectivity
resistance to inhibition
etc.
http://www.csa.com/discoveryguides/biofuel/review6.php
READ: http://www.novozymes.com/en/about-us/brochures/Documents/Enzymes_at_work.pdf
• One gram of soil contains more than 4,000 different
microorganisms.
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The search for the right enzyme is a highly automated process.
Huge robotic systems can simultaneously test a culture of
microorganisms on 12 different substrates such as starch, proteins
or fats. With the help of the robots, Novozymes' researchers can
scan thousands of microorganisms in just a few days.
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The final test involves scanning the enzymes under the precise
working conditions in which that enzyme will be used in the final
product.
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Data from: http://www.novozymes.com/en/about-us/our-business/what-areenzymes/Pages/finding-and-producing-enzymes.aspx
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Yellowstone hot springs
http://waynesword.palomar.edu/ploct97.htm
Deep sea thermal vents
http://microbes.nres.uiuc.edu/NRES512.htm
T. reesei growing
on a plate culture
Thermophilic bacteria isolated from Bag City Vent,
one of the vents that was part of the MBL/JISAO
microbial diversity study. (c) Julie Huber
http://www.chem.mq.edu.au/
edge/fungal-biotech.html
Clockwise from top left: Amanita muscaria, a basidiomycete; Sarcoscypha coccinea, an
ascomycete; bread covered in mold; a chytrid; an Aspergillus conidiophore.
http://www.answers.com/topic/fungus
From presentation of Maria Ciaramella
Vester et al. Microbial Cell Factories 2014, 13:72. Discovery of novel
enzymes with industrial potential from a cold and alkaline environment
by a combination of functional metagenomics and culturing
• The use of cold-active enzymes has many advantages,
including reduced energy consumption and easy
inactivation.
• The ikaite columns of SW Greenland are permanently
cold (4-6°C) and alkaline (above pH 10), and the
microorganisms living there and their enzymes are
adapted to these conditions.
• Since only a small fraction of the total microbial diversity
can be cultured in the laboratory, a combined approach
involving functional screening of a strain collection and a
metagenomic library was undertaken for discovery of
novel enzymes from the ikaite columns.
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Huge progress in genome sequencing
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Scientists needed $3 billion and 13 years to sequence
the three billion base pairs encoded in a single human
genome - the first time (completed in 2003).
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By 2011, eight years after that first project was
completed, the cost of sequencing a human genome
had fallen to $5,000, in a process that took just a few
weeks.
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And in January, Jonathan Rothberg, a chemical
engineer and the founder of the biotech company Ion
Torrent, unveiled an approach that is faster and
cheaper still. He says his machine will be able to
sequence a human genome, some 3.2 gigabytes’
worth of data, in two hours for just $1,000.
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http://www.popsci.com/science/article/2012-02/1000-genome-medicine-hasnew-problem-too-much-information
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http://www.nature.com/news/technology-the-1-000-genome-1.14901
>3000 genomes sequenced in 2011; > 18000 in 2014
Sequencing the genome creates so much data we don’t know what to do
with it.
Scientists are expecting as many as 1 billion people to have their genomes
sequenced by 2025.
Doolittle, 2002. Nature 419, 493-494
http://www.the-scientist.com/?articles.view/articleNo/39742/title/Sequencing-the-Tree-of-Life/
Structure of eukaryotic genes
Protein expression
http://genmed.yolasite.com/fundamentals-of-genetics.php
http://www.piercenet.com/browse.cfm?fldID=F46A98FE-C7DD-4868-AB2C-2E3C71FC84D1
DNA sequence –> amino acid sequence
Codon tables
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GenBank: 188 372 017 sequences in Oct 2015
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Cloning of genes
From www.explorebiology.com
http://www.blackwellpublishing.com/allison/docs/sample_ch8.pdf
Cloning of cDNA
using BamHI
linkers
From presentation of Linnea Fletcher BIOL 2316
http://www.ncbe.reading.ac.uk/ncbe/gmfood/chymosin.html
Chymosin protease is used in cheese production
to precipitate casein protein.
https://en.wikipedia.org/wiki/Chymosin
Gene libraries
http://www.emunix.emich.edu/~rwinning/genetics/tech2.htm
DNA libraries
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Genomic DNA Libraries - These libraries are made from genomic DNA (all of the
DNA found in the organism's nuclei). Genomic DNA molecules are very large (each
chromosome in the nucleus is one such DNA molecule), so they must be fragmented
into small enough pieces to insert into vectors. This is typically done through
digestion with one or more appropriate restriction endonucleases, mechanical
shearing, or a combination of the two processes. The DNA is then ligated into the
vector, which could be a plasmid, but is more often a cosmid or a viral chromosome.
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cDNA Libraries - These libraries are made from cDNA, which are DNA copies of
mRNA molecules. To make cDNA, mRNA is isolated from a tissue or whole
organism, and DNA is copied from the mRNA template using an enzyme called
reverse transcriptase. This enzyme works like a DNA polymerase, except that is
uses RNA as a template instead of DNA. The resulting cDNA molecules are then
engineered so that they have restriction enzyme recognition sites at each end of
every molecule, which allows them to be digested and inserted into a vector as
outlined previously.
http://www.emunix.emich.edu/~rwinning/genetics/tech2.htm
Metagenomic
libraries
http://muou.sc.mahidol.ac.th/research_an.html
cDNA Libraries are best for eukaryotes
No introns
in cDNA
From presentation of Linnea Fletcher BIOL 2316
Screening for specific
cDNA plasmids in a cDNA
Library by using an antibody probe
The antibody binds to a specific
Site on a protein that is made via
The inserted foreign DNA. This
Is a Western Blot since it uses antibody
To detect a protein.
From presentation of Linnea Fletcher BIOL 2316
High throughput screening
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Leemhuis et al. High-throughput screening for
gene libraries expressing carbohydrate
hydrolase activity. Biotechnology Letters 25:
1643–1645, 2003.
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A simple and fast method is described
allowing screening of large number of
Escherichia coli clones (4000 per day)
for the presence of functional or
improved carbohydrate hydrolase
enzymes. The procedure is relatively
cheap and has the advantage that
carbohydrate degrading activity can be
directly measured using liquid cultures
grown in microtiter plates without the
need of separation or purification steps.
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http://www.youtube.com/watch?v=Ap64ruCQTOQ
Screening for improved carbohydrate
hydrolase activity. Starch as substrate.
Increase of reducing sugars dtetcted.
http://www.exptec.com/Expression%20Technologies/Host%20cells.htm
Advantages of fungal expression systems
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High yield
Stable production strains
Cost effectiveness
High density growth
Easy scale-up
Safety
High expression levels
Rapid growth in chemically
defined media
Product processing similar to
mammalian cells
Can handle S–S rich proteins
Can assist protein folding
Can glycosylate protein
Sharma et al. World J Microbiol Biotechnol (2009) 25:2083–2094
Protein secretion: over 100 g/L
Milestones in protein engineering
Luetz et al., 2008
New Biocatalysts: biocatalysis roadmap 1999
http://www1.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf
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developing biocatalysts which are better, faster and cheaper than
current chemical catalysts
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development of a tool box of biocatalysts, i.e., biocatalysts that can
catalyze a broader range of reactions and have greater versatility than is
now possible
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increased temperature stability, activity, and solvent compatibility
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developing molecular modeling to permit rapid de novo design of new
enzymes
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creating better tools for new biocatalyst development
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educating the public to the societal benefits of using and creating
biocatalysts
New Biocatalysts: biocatalysis roadmap 1999
http://www1.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf
Engineering of Good Biocatalyst
Luetz et al., 2008
Protein Engineering Approaches
A) Random mutagenesis
– Error-prone PCR, Mutator strains
– Global
– Site specific (pick up some interesting sites)
B) DNA shuffling
– In vitro recombination between members in a protein family
– Methods have been developed for non-homologous recombination
 Recombination effectively exploits information present in the parental
sequences to form new functional sequences.
C) Directed evolution
– In each mutation round the best variants are selected and used for a new
round of mutagenesis or DNA shuffling.
– Screening method is critical for good result.
D) Phage-display
– A library can be made into bacteriophage genome
– The expressed proteins are exposed on the surface of the bacteriophage
– The bacteriophages can be screened by binding or enzymatic assays
– 109 variants screened
E) Rational design
– Rational design utilizes information on structure-function relationships
– Site-directed mutagenesis.
– Sequence comparison of thermophilic and mesophilic protein family
members (semi-rational design).
– Consensus sequences (semi-rational design).
– Computational methods
F) De novo design
– Artificial folded proteins.
– Protein scaffolds – new activities are engineered into them.
http://ocw.mit.edu/courses/biological-engineering/20-109-laboratory-fundamentals-in-biologicalengineering-spring-2010/lecture-notes/MIT20_109S10_lec_m2d2.pdf
http://ocw.mit.edu/terms/
Directed
evolution
Screening method
is critical. It determines
What will be obtained
by directed evolution
Gene shuffling
http://www.proteus.fr/en/integrated-platform/proprietary-technologies/protein-directed-evolution/
Quin, ACS Catal. 2011 Sep 2;1(9):1017-1021
Site-directed Mutagenesis Using PCR
1. Identify
site for
mutation
2. Denature
the plasmid
and anneal
primers
containing
the desired
mutation
3. Add DNA
polymerase
and extend
the primers
4. Use
Dpn1 to
degrade the
original
plasmid.
Some examples of rational design
- Structure-based design
Lutz. Curr Opin Biotechnol. 2010 December ; 21(6): 734–743.
B factor stabilization
1TE1
Reetz et al. Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for
Increasing Protein Thermostability. Angew. Chem. Int. Ed. 2006, 45, 7745 –7751
Böttcher and Bornscheuer. Current Opinion in Microbiology 2010, 13:274–282
From the presentation of Novozymes in NWBC, Helsinki 20.10.2015
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