Recent Advances In Enzymology

Addis Ababa University, College of Health

Sciences, Department of Biochemistry

By: Yohannes Gemechu( B.Sc., MSc. Fellow)

January 2015

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

Reference2

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 in

industrial processes.

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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

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.

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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.

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“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

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Techniques in Metagenomics

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.

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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

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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.

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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

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

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 :-

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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

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.

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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.

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2.2.4. Methods for protein engineering

A variety of methods are used in protein engineering:

Mutagenesis and selection

recombinant DNA technology.

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Proteins with Novel Properties

Rational Protein Design Nature

Random Mutagenesis

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

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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

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.

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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

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General strategy for

directed mutagenesis

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Requirements:

DNA of interest (gene or

promoter) must be cloned

Expression system must be

available -> for testing

phenotypic change

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 !!!

if methods efficient -> mostly mutated DNA

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Rational Protein Design

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Site –directed mutagenesis

Requirements:

Knowledge of sequence and preferable Structure

(active site,….)

Understanding of mechanism (knowledge about

structure – function relationship)

Identification of cofactors

Site-directed mutagenesis methods

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Old method

used before oligonucleotide

–directed mutagenesis

Limitations:

just C-> T mutations

randomly mutated

Site-directed mutagenesis methods

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Site-directed mutagenesis methods – Oligonucleotide

- directed method

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Site-directed mutagenesis methods – PCR

based

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2. Directed Evolution – Random mutagenesis

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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

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General Directed Evolution

Procedure

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Random mutagenesis methods

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

process (StEP)

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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

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Random Mutagenesis (PCR based) with degenerated

primers (saturation mutagenesis)

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Random Mutagenesis (PCR based)

with degenerated primers (saturation mutagenesis)

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Random Mutagenesis (PCR based)

Error –prone PCR

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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)

What can be engineered in Proteins ?

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Folding (+Structure):

1. Thermodynamic Stability

(Equilibrium between: Native Unfolded state)

2. Thermal and Environmental Stability (Temperature,

pH, Solvent, Detergents, Salt …..)

What can be engineered in Proteins ?

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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

Protein Engineering

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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)

Protein Engineering - Applications

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Engineering Stability of Enzymes – T4 lysozyme

S-S bonds introduction

Protein/Enzyme Engineering - Applications

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Engineering Stability of Enzymes – triose phosphate

isomerase from yeast

replace Asn (deaminated at high temperature)

Protein Engineering – Applications Cont’d

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Engineering Activity of Enzymes – tyrosyl-tRNA

synthetase from B. stearothermophilus

-> replace Thr 51 (improve affinity for ATP) -> Design

Protein/Enzyme Engineering - Applications

Directed Evolution

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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

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.

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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.

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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

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.

http://www.slideshare.net/

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