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662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis

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Title: 662 431 Molecular Biotechnology Application of molecular biotechnology in biocatalysis


1
662 431 Molecular Biotechnology Application of
molecular biotechnology in biocatalysis
  • ?. ??. ???? ??????????

2
Advantages of enzymes as biocatalysts
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  • ??????????????????????
  • ????????????????
  • ??????????????????? pH 2-12
  • ??????????????????????????????????????????? ??
    byproducts ?????????
  • ???????????????? (??????????????????)
  • ????????????????????????
  • ??????????????????????

3
Disadvantages of enzymes as biocatalysts
  • ????????????????????????????????????, pH
    ?????????????????? ??????????????????????????
  • ??????????????????????????????????????????????????
    ???
  • ?????????????????? proteases
  • ??????????????????????????

4
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

5
Applications of enzyme engineering
  • Improving enzyme activity
  • Changing enzyme substrate specificity and
    selectivity
  • Enhancing enzyme stability
  • Altering enzyme mechanism

6
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

7
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.

8
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
9
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.

10
Genetic code
11
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.

12
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

13
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.

14
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

15
Directed evolution (III)
  • The ultimate goal of directed evolution is to
    accumulate improvements through repetitive rounds
    of mutagenesis and identification.

16
Directed evolution (IV)
17
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

18
Error-Prone PCR
  • (A) gene amplification under error triggering
    conditions
  • (B) set of mutated gene fragments
  • (C) cloning into an expression vector

19
PCR ep-PCR
20
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

21
Gene Shuffling
  • DNAse I fragmentation of parental genes
  • assembly of recombined
  • genes using outer primers
  • (C) cloning into an expression vector.

22
Directed evolution of some enzymes
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