Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”

1. Recent Advances In Enzymology
Addis Ababa University, College of Health
Sciences, Department of Biochemistry
By: Yohannes Gemechu( B.Sc., MSc. Fellow)
January 2015

2. Outline
Introduction
Advances in Enzymology
Metagenomics
Protein/Enzyme Engineering
Mutagenesis
Site Directed Mutagenesis
Random Mutagenesis
Chemical Modification of Enzyme
De novo synthesis of modified gene
Summary
Reference 2

3. 1. Introduction
 Enzymes are natural catalysts.
 They are produced by living organisms to increase the rate of
an immense and diverse set of chemical reactions required for
life.
 They are involved in all processes essential for life such as
DNA replication and transcription, protein synthesis,
metabolism and signal transduction, etc.
 And their ability to perform very specific chemical
transformations has made them increasingly useful in3

4. 2. Advances in Enzymology
 Many worldwide corporations have recognized the bio-
based technologies as one of the key drivers of sustainable
growth.
 However, the biological process is often considered only
when the chemical arsenal has failed to achieve synthesis
of the target molecule.
 This is primarily because the unavailability of the desired
enzyme to catalyze the reaction in an efficient manner.
 The exploitation of new types of enzymes, improvements
of enzyme properties and of the production process are
overall goals of innovation in the enzyme manufacturing
industry.
4

5. 2. Advances in Enzymology cont’d
 Systematic methods in the field of enzyme and reaction
engineering have allowed access to means to achieve the
ends, i.e.
 Screening for novel enzymes from natural samples
with improved characteristics(Metagenomics)
 Engineering the existing enzymes using genetic
engineering approaches (Protein/Enzyme
Engineering),
 Fining the enzyme processes in the enzyme
manipulation to overcome catalyst limitation,
e.g. downstream processing in enzyme manufacturing,
formulation of enzyme preparations and enzyme
immobilization, etc.
5

6. 2.1. Metagenomics
 Study of metagenome (genomic content of entire microbial
community), genetic material recovered directly
from environmental samples.
 Also referred as Environmental genomics, Ecogenomics, or
community genomics.
 The term "metagenomics" was first used by Jo Handelsmann,
Jon Clardy, Robert M. Goodman,and others, and first appeared
in publication in 1998.
6
“The application of modern genomics techniques to the
study of communities of microbial organisms directly in
their natural environments, bypassing the need for
isolation and lab cultivation of individual species”
- Kevin Chen and Lior Pachter

7. 7
Techniques in Metagenomics

8. TWO APPROACHES FOR METAGENOMICS
 In the first approach, known as
‘sequence-driven
metagenomics’, DNA from the
environment of interest is
sequenced and subjected to
computational analysis.
 The metagenomic sequences are
compared to sequences
deposited in publicly available
databases such as GENBANK.
 The genes are then collected into
groups of similar predicted
function, and the distribution of
various functions and types of
proteins that conduct those
functions can be assessed.
 In the second approach,
‘function-driven
metagenomics’, the DNA
extracted from the
environment is also
captured and stored in a
surrogate host, but instead
of sequencing it, scientists
screen the captured
fragments of DNA, or
‘clones’, for a certain
function.
 The function must be absent
in the surrogate host so that
acquisition of the function
can be attributed to the metagenomic
DNA.
8

9. Novel enzyme production using
Metagenomics library
 Chitinase production from Marine environment
 Screening gene coding for chitin degrading enzyme by using
analogues (4-methylumbeliferyl-D-N,N’-diacetylchitobioside
(MUF-diNAG)
 MUF-diNAG flourogenic chitin analog
 9 positive clones from 750,000 sample
9

10. 2.2. Protein/Enzyme Engineering
 Protein engineering can be defined as the modification
of protein structure with recombinant DNA technology
or chemical treatment to get a desirable function for
better use in medicine, industry and agriculture.
10

11. 2.2.1. Objectives of Protein/Enzyme Engineering
 The objectives of protein engineering is:
 to create a superior enzyme to catalyze the production
of high value specific chemicals.
 to produce enzyme in large quantities.
 to produce biological compounds(include synthetic
peptide, storage protein, and synthetic drugs) superior
to natural one. 11

12. 2.2.2. Rationale of Protein Engineering
 For industrial application an enzyme, should possess some
characteristics in addition to those of enzymes in cells.
 These characteristics are :-
 enzyme should be robust with long life.
 enzyme should be able to use the substrate supplied in
the industry even it differs from that in the cell.
 enzyme should be able to work under conditions, e.g.
extreme of pH, temperature and concentration of the
industry even if they differ from those in the cell.12

13. Rationale of Protein Engineering cont’d
 In view of above, the enzyme should be engineered to
meet the altered needs.
 Therefore, efforts have been made to alter the properties
of enzymes.
 Characters that one might have to change in a
predictable manner in enzyme engineering to get the
desired function :-
13

14.  Kinetic properties of enzyme-turnover and
Michaelis constant, Km.
 Thermo stability and the optimum temperature
for the enzyme.
 Stability and activity of enzyme in nonaqueous
solvents.
 Substrate and reaction specificity.
 Cofactor requirements
 Optimum PH.
 Molecular weight and subunit structure.
14
Rationale of Protein Engineering cont’d

15.  Therefore for a particular class of enzymes, variation in
nature may occur for each of the above properties, so
that one may like to combine all the optimum properties
to the most efficient form of the enzyme.
 For e.g. glucose isomerases, which convert glucose into
other isomers like fructose and are used to make high
fructose corn syrup vital for soft drink industries.
15

16. 2.2.3. Basic assumption for protein
engineering While doing protein engineering should recognize the
following properties of enzymes:
 many amino acid substitution, deletions or additions lead
to no changes in enzyme activity so that they are silent
mutator.
 Protein have limited number of basic structures and only
minor changes are superimposed on them leading to
variation
 Similar patterns of chain folding and domain structure can
arise from different amino acid sequences with little or no
homology.
16

17. 2.2.4. Methods for protein engineering
 A variety of methods are used in protein engineering:
Mutagenesis and selection
recombinant DNA technology.
17
Proteins with Novel Properties
Rational Protein Design Nature
Random Mutagenesis

18. 2.2.4.1. Mutagenesis
 Mutagenesis refers to a change in DNA sequence
 Point mutations or large modifications
 Point mutations (directed mutagenesis):
 Substitution: change of one nucleotide (i.e. A-> C)
 Insertion: gaining additional nucleotide
 Deletion: loss of nucleotide
18

19. Mutagenesis cont’d
 Mutagenesis and selection can be effectively utilized for
improving a specific property of an enzyme.
 E.g. E.coli anthranilate synthetase enzyme is normally
sensitive to tryptophan inhibitor due to feedback
inhibition but an altered MTR2 mutation of E.coli was
found to possess an altered form of enzyme anthranilate
synthetase that is insensitive to tryptophan inhibition.
 And thus helping in the continuous synthesis of
tryptophan without inhibition.
19

20. Mutagenesis cont’d
Mutagenesis can lead to gene modification.
The two ways of gene modification are -
(a) In vitro mutagenesis using synthetic oligonucleotides.
(b) De novo Synthesis of complete modified gene.
20

21. In vitro mutagenesis using synthetic oligonucleotides.
 Synthetic oligonucleotides is used for invitro
mutagenesis.
 In this method, a small oligonucleotides primer
containing the desired modification is first synthesized.
 It is then hybridized to the appropriate site and cloned
gene and then the rest is replicated using DNA
polymerase enzyme, so that the rest remains unaltered.
 This approach is actually used to modify the active site
of the tyrosyl-tRNA synthetase
21

22. General strategy for
directed mutagenesis
22
Requirements:
 DNA of interest (gene or
promoter) must be cloned
 Expression system must be
available -> for testing
phenotypic change

23. Approaches for directed mutagenesis
1. Site-directed mutagenesis
 point mutations in particular known area
Give rise to library of wild-type and mutated DNA
(site-specific)
not really a library -> just 2 species
2. Random mutagenesis
 point mutations in all areas within DNA of interest
Give rise to library of wild-type and mutated DNA
(random)
a real library -> many variants -> screening !!!23

24. Rational Protein Design
24
 Site –directed mutagenesis
 Requirements:
 Knowledge of sequence and preferable Structure
(active site,….)
 Understanding of mechanism (knowledge about
structure – function relationship)
 Identification of cofactors

25. Site-directed mutagenesis methods
25
Old method
 used before oligonucleotide
–directed mutagenesis
Limitations:
 just C-> T mutations
 randomly mutated

26. Site-directed mutagenesis methods
26

27. Site-directed mutagenesis methods – Oligonucleotide
- directed method
27

28. Site-directed mutagenesis methods – PCR
based
28

29. 2. Directed Evolution – Random mutagenesis
29
 Based on the process of natural evolution
 NO structural information required
 NO understanding of the mechanism required
General Procedure:
 Generation of genetic diversity
 Random mutagenesis
 Identification of successful variants
 Screening and seletion

30. 30

31. General Directed Evolution
Procedure
31
Random mutagenesis methods

32. Evolutionary Methods
 Non-recombinative methods:
 Oligonucleotide Directed Mutagenesis (saturation
mutagenesis)
 Chemical Mutagenesis, Bacterial Mutator Strains
 Error-prone PCR
 Recombinative methods -> Mimic nature’s recombination
strategy
Used for: Elimination of neutral and deleterious mutations
 DNA shuffling
 Invivo Recombination (Yeast)
 Random priming recombination, Staggered extention
32

33. Evolutionary Methods
Type of mutation – Fitness of mutants
 Type of mutations:
Beneficial mutations (good)
Neutral mutations
Deleterious mutations (bad)
 Beneficial mutations are diluted with neutral and
deleterious ones
 Keeping the number of mutations low per cycle improve
fitness of mutants
33

34. Random Mutagenesis (PCR based) with degenerated
primers (saturation mutagenesis)
34

35. Random Mutagenesis (PCR based)
with degenerated primers (saturation mutagenesis)
35

36. Random Mutagenesis (PCR based)
Error –prone PCR
36
Use of PCR with low fidelity !!!
Achieved by:
 Increased Mg2+ concentration
 Addition of Mn2+
 Adding unequal concentration of
the four dNTPs
 Use of dITP
 Increasing amount of Taq DNA
polymerase (Polymerase with
NO proof reading function)

37. What can be engineered in Proteins ?
37
 Folding (+Structure):
1. Thermodynamic Stability
(Equilibrium between: Native  Unfolded state)
2. Thermal and Environmental Stability (Temperature,
pH, Solvent, Detergents, Salt …..)

38. What can be engineered in Proteins ?
38
 Function:
1. Binding (Interaction of a protein with its surroundings)
How many points are required to bind a molecule with high
affinity?
2. Catalysis (a different form of binding – binding the
transition state of a chemical reaction)
 Increased binding to the transition state  increased
catalytic rates .
 Requires: Knowledge of the Catalytic Mechanism.
-> engineer Kcat and Km

39. Protein Engineering
39
Factors which contribute to stability:
1. Hydrophobicity (hydrophobic core)
2. Electrostatic Interactions:
-> Salt Bridges
-> Hydrogen Bonds
-> Dipole Interactions
3. Disulfide Bridges
4. Metal Binding (Metal chelating site)

40. Protein Engineering - Applications
40
 Engineering Stability of Enzymes – T4 lysozyme
 S-S bonds introduction

41. Protein/Enzyme Engineering - Applications
41
 Engineering Stability of Enzymes – triose phosphate
isomerase from yeast
 replace Asn (deaminated at high temperature)

42. Protein Engineering – Applications Cont’d
42
 Engineering Activity of Enzymes – tyrosyl-tRNA
synthetase from B. stearothermophilus
-> replace Thr 51 (improve affinity for ATP) -> Design

43. Protein/Enzyme Engineering - Applications
Directed Evolution
43

44. 2.4.2. Chemical modification of enzymes
 The protein synthesized under the control of gene
sequence in a cell undergo post-transitional
modification.
 This leads to stability, structural integrity, altered
solubility and viscosity of individual proteins.
E.g:Enzyme-PEG conjugates.
 An enzyme L- asparaginase has anti-tumour properties
but is toxic with a life time of less than 18hrs thus
reducing its utility.
44

45. Chemical modification of enzymes
 PEG-L-asparginase conjugates differ from the
native enzyme in the following way:
it retains only 52% of the catalytic activity of
the native,
it become resistant to proteolytic degradation,
it doesn’t cause allergy.
45

46. De Novo Synthesis of Complete Modified Gene
 Complete gene in some cases have been chemically
synthesized in the form of several oligomers (e.g. genes
for insulin, somatostain and interferon), that are ligated
in correct order to produce a complete gene.
 The sequence of the synthetic gene can be designed in a
modular fashion to get the desired function.
46

47. Summary and Outlook
 In the past decades, many chemical industry were restrained
from embracing enzyme technology, largely because enzymes
were considered as being too delicate to survive the extreme
conditions in real reaction vessels.
 Some of the strategies in the field are exploiting novel enzymes
from nature, improving existing catalytic properties,
broadening specialized enzymes to serve new functions,
optimizing formulation of enzyme preparations, or de novo
designing biocatalysts.
 These approaches have provided valuable candidates for the
bio-catalytic processes.
 However, breakthroughs of enzyme products for biochemical
technology should be recruited.
47

48. References
 Li X., Zhang Z. and Song J. (2012) Computational enzyme
design approaches with significant biological outcomes:
progress and challenges. Comp and Struc Biotech Jour;2:3.
 Li SH., Yang X ., Yang SH., Zhu M . and Wang X. (2012)
Technology Prospecting on Enzymes: Application,
Marketing and Engineering. Comp and Struc Biotech
Jour;2:3.
 Handelsman J. (2004 )Metagenomics: Application of
Genomics to Uncultured Microorganisms. Microbiol Mol
Biol Rev; 68(4): 669–685. 48

49. 49