662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis

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662 431 Molecular Biotechnology Application of
molecular biotechnology in biocatalysis

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Advantages of enzymes as biocatalysts

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Disadvantages of enzymes as biocatalysts

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Approaches to engineering enzyme activity

• Rational protein design
• (computer-aided molecular modeling and
  site-directed mutagenesis)
• Directed evolution
• (random mutagenesis / recombination and
  screening / selection method)
• Semi-rational protein design

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Applications of enzyme engineering

• Improving enzyme activity
• Changing enzyme substrate specificity and
  selectivity
• Enhancing enzyme stability
• Altering enzyme mechanism

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Rational protein design (I)

• Usually requires both the availability of the
  structure of the enzyme and knowledge about the
  relationships between sequence, structure and
  mechanism
• Using molecular modeling, it has been possible to
  predict how to increase the selectivity, activity
  and the stability of enzymes

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Rational protein design (II)

• Amino acid substitutions are often selected by
  sequence comparison with homologous sequences.
• Comparison of the three-dimensional structures of
  mutant and wild-type enzymes are necessary to
  ensure that a single mutation is really
  site-directed.

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Rational protein design (III)
Protein structure
Planning of mutants Site-directed mutagenesis
Vector containing mutated genes
Transformation in E. coli
Protein expression purification
Mutant enzymes
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Rational Design Using Site-Directed Mutagenesis
(I)

• Saturation mutagenesis is basically a
  site-directed mutagenesis protocol adapted to the
  use of degenerate oligonucleotides (NNN or NNK
  mutagenic cassettes, with N A, T, G, C and K
  G, T for instance) to introduce a full diversity
  (the 20 amino acids) at a given position.

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Genetic code
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Rational Design Using Site-Directed Mutagenesis
(II)

• degenerated codons introduced by PCR
• (B) overlap PCR assembly
• (C) set of degenerated gene fragments
• (D) cloning into an expression vector.

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Examples of enhance thermostability

• The removal of asparagine residues in a-amylase
• The introduction of more rigid structural
  elements such as proline into a-amylase and
  D-xylose isomerase
• Addition of disulfide bridges to stabilize hen
  lysozyme

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Directed evolution (I)

• It mimics the process of Darwinian evolution in
  the test tube, combining mutagenesis and
  recombination with selection or screening for
  improved variants with the desired
  characteristics.
• The main advantage is that the enzymes
  properties and functions can easily be engineered
  even without any knowledge of the structure.

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Directed evolution (II)

• Random mutagenesis of the gene encoding the
  catalyst or recombination of gene fragments
• The variants are analysed on the basis of the
  properties of interest by either screening or
  selection.
• The gene(s) encoding the improved variants are
  identified and then used to parent the next round
  of directed evolution

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Directed evolution (III)

• The ultimate goal of directed evolution is to
  accumulate improvements through repetitive rounds
  of mutagenesis and identification.

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Directed evolution (IV)
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Random Mutagenesis Using Error-Prone PCR

• A starting gene is amplified over a million fold
  in an imperfect copying process that generates
  uncontrolled errors.
• The technique is a variation of standard PCR
  using unbalanced deoxyribo-nucleotides
  concentrations, high Mg2 concentration, Mn2,
  low annealing temperatures, or a high number of
  cycles which are all error-triggering factors

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Error-Prone PCR

• (A) gene amplification under error triggering
  conditions
• (B) set of mutated gene fragments
• (C) cloning into an expression vector

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PCR ep-PCR
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Recombination of gene fragment using Gene
Shuffling

• The recombination of homologous genes harvested
  from nature.
• the parental genes have been preselected by
  natural evolution as functional hence their
  progeny has a good chance of containing improved
  genes due to additive or synergistic
  combinations.
• Fragmentation step of the parental genes followed
  by the random reassembly of parental gene segments

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Gene Shuffling

• DNAse I fragmentation of parental genes
• assembly of recombined
• genes using outer primers
• (C) cloning into an expression vector.

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Directed evolution of some enzymes