enzyme regulation as industrial strain improvement technology

1. ENZYME REGULATIONS AS
AN INDUSTRIAL STRAIN
IMPROVEMENT STRATEGY
NAME : ZEENAT JAHAN
CLASS : MSC. (AGRI ) MICROBIOLOGY 3 RD SEMESTER
SUBMITTED TO : PROF. IQBAL AHMAD
DATE OF SUBMISSION : 5/09/2020
PAPER CODE : MBM- 3008
ENROLLMENT NUMBER : GL5194

2. CONTENTS ;
1. Strain & it’s definition
2. Strain improvement and it’s techniques
3. Enzyme regulations
1. Enzyme activity
2. Enzyme Synthesis
4. Mechanism of enzyme regulations
1. Regulation of enzyme activity
2. Regulation of enzyme availability
5. Common mechanism of Enzyme regulations for strain
improvement and it’s significance

3. STRAIN IT’S DEFINITION AND EXAMPLES
1. Strain is a genetic variant or subtype of a microorganisms (
eg. Virus or bacterium or fungus ). For example “ flu strain” is
certain form of flu virus which causes influenza .
2. It is the simplest level of classification. All organism
belonging to the same strain are exactly same without any
difference. For example Escherichia coli BL21(DE3) ,
Escherichia coli B etc.

4. STRAIN IMPROVEMENT AND IT’S
TECHNIQUES
• Strain improvement by mutagenesis and by other alternative
methods is a highly developed technique which plays a critical role
in the commercial development of Microbial fermentation processes
. This importance of Microbial Genetics was realised during 1940s
when the global bloom for Penicillin production started.
• There are some of the Strain improvement technology discussed as
follows ;
continue.

5. STRAIN IMPROVEMENT TECHNOLOGIES
Mutant selection Genome
Shuffling
Recombination Protoplast fusion RecombinationD
NA technology
Mutation has
increased the
productivity of
industrial
cultures. It has
also helped to
increase the
metabolite
production in the
fermentation
broth as well as
the production
of secondary
metabolites is
also eased . The
most common
method is to
Genome
Shuffling is
widely used for
increasing the
production of
metabolism by
bacterial strains,
improving
substrate uptake
as well as
enchancing
strain tolerance.
It is a combined
technique which
can be used as
multiplication
crossing allowed
ThIs method of
strain
improvement is
not much
common
because of
extremely low
frequency of
Genetic
recombination in
industrial
microorganisms
,for ex:
Streptmyces it
was 10 -6 or
even less. It was
erroneously
Development of
this technology
has changed the
mindset for
recombination.
The method was
highlighted and
preferred for the
production of
Microbial
product like
Antibiotics . It
has been applied
for the fusion
between
different mutant
line.
Recombinant
DNA technology
refers to the
joining together
of DNA molecule
from two
different species
that are inserted
into a host
organism to
produce new
genetic
combination.
Also called as
rDNA
technology.
It was first

6. ENZYME REGULATIONS
• Enzyme regulations is control of the rate of a reaction catalyzed
by an enzyme , by some effector (e.g. initiators or activators )
or by alterations of some conditions (e.g. pH or ionic strength )
.
• There are two levels of enzyme regulations ;
1. Enzyme Activity
2. Enzymes Synthesis

7. DEFINITION
• Enzyme Activity
• It is the catalytic effect exerted by an enzyme, expressed as units per
milligrams of enzyme (specific enzyme ) or molecules of substrate
transformed per minute per molecule of enzyme (molecular activity) .

8. CONTINUE ..
2. Enzyme Synthesis :
• Enzyme are best known as biological catalyst therefore are synthesized
in all living cells. As we know most of the enzymes are protein in nature
and the protein is made of polypeptide chains which in turn is made of
small subunits called amino acids and this polypeptide are synthesized
by a process called “ translation.”.

9. MECHANISM OF ENZYMES REGULATIONS
• The mechanism of enzymes regulations can be studied under
two headings :
• Regulation of enzyme activity
• Regulation of enzyme availability or quality

10. REGULATION OF ENZYME ACTIVITY
• Enzyme Activity must be regulated so that the proper levels or
products are produced at all times and places.
• Catalytic activity is affected by following mechanisms;
1. Allosteric regulation
2. Covalent modification
1. Regulation by reversible covalent modification
2. Regulation by irreversible covalent modification
3. Regulation by protein protein interaction
4. Feedback inhibition
5. Proenzymes

11. REGULATION OF ENZYME AVAILABILITY OR
QUANTITY
1. Regulation of enzyme Synthesis
2. Regulation of enzyme degradation
3. Compartmentalization of enzyme activity
4. Differential activity of isozymes
5. Induction
6. Repression

12. COMMON MECHANISM OF ENZYME
REGULATIONS FOR STRAIN IMPROVEMENT AND
IT’S SIGNIFICANCE
1. Enzyme activity
1. Allosteric regulation
Enzyme action must be regulated so that in a given cell at a given time ,
the desired reaction are being catalyzed and the undesired reaction are
not . Inhibition and activation of enzymes via other molecules are
important ways that enzyme are regulated. Inhibition activity is
competitive and non competitive . Non competitive inhibition are usually
Allosteric (allo – other ) and ( steric – form ) regulation.

13. SIGNIFICANCE OF ALLOSTERIC REGULATION
• Allosterically regulated enzyme is essential to develop a highly efficient
metabolites production.
• To design process of allosterically controllable enzymes, we develop an
effective competitional strategy to deregulate the allosteric inhibition of
enzyme based on sequence evolution analysis of all ligand – ligand sites .
The allosteric sites are comprised of more hydrophobic residues than
catalytic sites .
• For example –
• the productivity of ethanol formation process is dramatically increased by a
mutation in pyruvate dehydro complex which leads to the complex being less
sensitive to the allosteric inhibition by NADH .

14. LYSINE PRODUCTION
• large scale production of l- lysine with C. glutamium started as
early as 1958 at Kyowa Hakko’s Plant in Japan .
• In short , the flux through the split lysine biosynthesis pathway
was increased by over expression for the genes for the key
ASPARTATE ( oxaloacetate – derived & catalyzed by
aspartokinaseLysin
e
Methionin
e
Threoni
ne

15. MECHANISM OF ALLOSTERIC REGULATION
• Enzyme have an area called the active site, where they bind
substrates .
• Many enzymes also have other areas called allosteric sites, located
in different place from active site .
• An Allosteric sites does not bind substrate, but instead binds
another molecule that affects the enzyme’s regulation.
• Allosteric enzyme regulation is when a molecule binds a site
othertha the active site & changes the behaviour of enzyme by
changing it’s conformation .
• In most cases the binding allosteric sites acts like a dimer switch
which can activate allosteric molecule & turn up enzyme activity .
• They can also lower , inactivate and turn off the enzyme .
• Activation state of an enzyme is referred as ‘R’ / relaxed state ( in

16. CONTINUE…
• T= tense state , enzyme off and activity turned down.
• One molecule binding to the allosteric sites and make the enzyme change
from the T to R state while some can do opposite .
• For example – phosphofructokinase -1 is activated by ADP but inactivated
by ATP.
• PFK1 inhibition by metabolites
intermediates such
as citrate , lactate & ATP results in
increase flux
through the PPP .
PKK2 activation by AMP , ADP or

17. KINETICS OF ALLOSTERIC ENZYME
• Allosteric enzyme are an exception to the Michaelis – Menten
model . Because they have more than two subunits and active
sites , the do not obey the Michaelis- Menten kinetics but
instead have sigmoidal kinetics . Since allosteric enzyme are
cooperative or sigmoidal plot results.

18. FEEDBACK REGULATION
• The most important mechanism responsible for regulation of the enzyme
involved in biosynthesis of amino acids , nucleotides and vitamins is not
induction or nutrient repression but feedback regulation . This category of
regulation function at two levels :
• Enzyme Action ( feedback inhibition)
• Enzyme Synthesis ( feedback repression & attenuation)
FEEDBACK INHIBITION
• The final metabolism of a pathway, when present in sufficient quantities , inhibits the
action of the first enzyme of the pathway to prevent further , synthesis of intermediates
& products of that pathway.
• It is controlled by
• Non competitive inhibition &
• Competetive inhibition
• Non competitive inhibition – the inhibition is end product of metabolic pathway that is
able to bind to a second site (allosteric sites ) on the enzyme. Binding of the inhibitor to
the allosteric site alters the shape of the enzyme active site thus preventing binding of

19. NON COMPETITIVE INHIBITION OF ENZYME
ACTIVITY

20. COMPETITIVE INHIBITION OF ENZYME
ACTIVITY .
THE END PRODUCT ( INHIBITOR ) OF A PATHWAY BINDS TO THE ACTIVE SITE OF THE FIRST ENZYME IN THE
PATHWAY. AS A RESULT, THE ENZYME CAN NO LONGER BIND TO THE STARTING SUBSTRATE OF THE PATHWAY.

21. SIGNIFICANCE OF FEEDBACK INHIBITION
• Allosteric regulation of
phosphoenolpyruvate carboxylase
(PEPC) control the metabolic flux
distribution of anaplerotic pathway.
• The feed back inhibition of
Corynebacterium glutamium PEPC
was rationally deregulated & it’s
effect on metabolic flux
redistribution was evaluated.
• On this basis 6 PEPC mutants were
designed and all of them showed
reduced sensitivity towards
ASPARTATE and MALATE inhibition.
Introducing one of the point
mutation into ppc gene , encoding
PEPC of the lysine producing strain
Corynebacterium glutamium LC298
• Allosteric regulation of the central
metabolism and lysine synthesis
pathways in C. glutamicum. Feedback
inhibitions of PEPC by aspartate and
malate and AK by threonine and lysine
strictly control the metabolic flux to
lysine synthesis pathway. Dotted arrows
represent pathways consisting of
several reactions. PC, pyruvate
carboxylase, PEPCK,

22. ENZYME SYNTHESIS ( FEEDBACK
REPRESSION & ATTENUATION)
• Feedback repression
• Feedback repression involves the
turning off of enzyme synthesis when
sufficient amounts of the product
have been made & it starts to
accumulate . The end product of the
pathway acts as a co- repressor , an
active repressor is formed which
binds to the operator to prevent the
transcription by RNA POLYMERASE &
hence prevents enzyme synthesis.
• Feedback attenuation
• Many of the amino acids biosynthesis
pathway are not regulated by amino
acids themselves but by their charged
tRNA molecule. Thus , whereas
feedback repression is effected by
aminoacids and end products acting
as co represso interfering with
transcription initiation , another type
of control called attenuation (
Transcription terminate or control )
involves charged tRNA & transcript
termination.
• Example : it is known to control
certain bacterial aminoacid
biosynthetic operons like threonine,
isoleucine,valine phenylalanine,
histidine .

23. PROENZYME OR ZYMOGEN
• Proenzyme also known as zymogen.
• Proenzyme is a any group of protein that
displays no catalytic activity but are
transformed within an organism into
enzyme , especially those that catalyze
reactions involving the breakdown of
proteins.
• For example , Trysinogen and
chymotrypsinogen are zymogens secreted
by Pancreas are activated by the instestinal
tract to Trypsin and chymotrypsin .
• Activation is effected by the cleavage of one
or more peptide bonds of the zymogen
molecule and may be catalyzed by a
separate enzyme.
• For example , enterokinase converts

24. BIOLOGICAL SIGNIFICANCE OF ZYMOGENS
• Digestive Enzymes
• Ex , Pepsinogen it is an inactive protein which
is cleaved in the stomach to produce pepsin a
digestive enzyme .
• Protein hormones – some are activated in
inactivated forms of the actual enzyme.
• Ex , A prime example is insulin. Insulin arises
from an inactive form known as proinsulin. It is
activated by proteolytic cleavage of a specific
peptide. Proinsulin is first synthesized in the
endoplasmic reticulum where the peptide chain
is folded and the disulfide bonds oxidized. It is
then packaged in the Golgi Apparatus and it is
also proteolyically cleaved by series of
proteases to form insulin. The matured insulin
has 39 less amino acids than the proinsulin: 4
are removed and recycled and the remaining
35 amino acids form the C-peptide .

25. COVALENT MODIFICATION
• Regulation inactivation refers
to the selective inactivation of
enzyme (Switzer,1977) by two
different mechanisms.
• In modification inactivation
the enzyme remains intact
but it’s physical state is
changed or is covalently
modified.
• The covalent modification can
be studied under two
headings :
1. Reversible covalent
modification
2. Irreversible covalent
modification

26. REVERSIBLE AND IRREVERSIBLE COVALENT
MODIFICATIONS
• Reversible covalent modification
• Reversible covalent modification is typically
associated with the regulation of signalling and
metabolic processes and select critical target’s
can be modified by multiple types of
modification of aminoacids with multiple
number of modification.
• Reversible modification is typically associated
with regulatory processes or in regulatory steps
of metabolic and signalling pathways.
• Some of the reversible covalent modification are
–
1. Methylation – methylation of glutamate or
aspartate carboxyl group.
2. Phosphorylation – phosphorylation of specific
serine or threonine residue,nucleotidulation of
specific tyrosine residue , ADP ribosylation of an
arginine residue .
3. Acetylation – acetylation of amino group of
lysine residue or tyrosinalation of a protein

27. 2. IRREVERSIBLE
COVALENT
MODIFICATION
• Irreversible covalent
modification
• Irreversible modification are
more energetically costly
requiring the synthesis of new
protein before the fundamental
modification can be
accomplished if needed.
• Irreversible modification can be
seen with the physiological
cascade processes such as
blood coagulation.
• Irreversible modification
• For example –

28. REGULATION OF PROTEIN- PROTEIN
INTERACTION• Proteins control all biological systems in
a cell , and while many proteins perform
their functions independently .
• The protein expression is a dynamic
process, the protein that are used to
complete specific tasks may not be
always expressed or activated .
• Protein interactions are fundamentally
characterized as stable or transient, and
both types of interactions can be either
weak or strong.
• For example , haemoglobin and core
RNA polymerase are examples of multi
subunit interactions that form stable
complexes.
• Transient interactions are expected
to control the majority of cellular
processes. As the name implies,
transient interactions are temporary
in nature and typically require a set
of conditions that promote the
interaction, such as
phosphorylation, conformational
changes or localization to discrete
areas of the cell .
• Transiently interacting proteins are
involved in a wide range of cellular
processes, including protein
modification, transport, folding,
signaling, apoptosis and cell
cycling. The following example
provides an illustration of protein
interactions that regulate apoptotic

29. CONTINUE….
• Recently, scientific efforts have helped
towards the understanding and the
deciphering of the protein-interaction
networks (PINs) that forge the bacterial
interactome .However, despite the potential
of these bacterial PPI maps, they have only
been studied in detail in a few
microorganisms including Escherichia coli
(one of the best-studied model organisms in
this field) , Mycobacterium tuberculosis ,
Helicobacter pylori Pseudomonas aeruginosa
, Campylobacter jejuni Treponema pallidum
& the Cyanobacterium Synechocystis spp. ,
Mesorhizobium , Mycoplasma pneumoniae .
Furthermore, partial PINs for Bacillus subtilis
and Streptococcus pneumoniae have been
reported recently, and many more are near
completion .

30. REGULATION OF ENZYME AVAILABILITY OR
QUALITY
1. Regulation of enzyme synthesis
Enzymes can be controlled and
regulated in two ways :
1. Controlling the synthesis of enzyme
2. Controlling the activity of enzyme
OR
They function as –
1. Repressors /repression
2. Inducers / induction
• Repression , repression in metabolism in
which a protein molecule , called repressor ,
prevents the synthesis of an enzyme by
binding to thereby deoxyribonucleic acid that
controls the process by which the enzyme is
synthesized.
• Example for repression – in L – glutamic acid
production , normally the glutamic acid
overproduction is not expected to occur due to
feedback inhibition. Glutamate feedback
controls include repression of PEP carboxylase,
citrate synthase and NADP-GDP ; However, by
decreasing the effectiveness of the cell barrier
to outward passage, glutamate can be pumped
out of the cell thus allowing it’s biosynthesis to
proceed unabated.
• a deregulated strain of E.coli in which feedback

31. INDUCTION
• This is a control mechanism by which
a substrate ( orbs compound
structurally similar to the substrate or
a metabolically related compounds) ‘
turns on’ the synthesis of enzymes,
which are usually involved in the
degradation of the substrate.
• Enzymes that are synthesized as a
result of genes being turned on are
called inducible enzymes and the
chemical is called that activates gene
transcription is called the inducer .
• For example : the most thoroughly
studied inducible enzyme system is
that for lactose hydrolysis in E.coli ,
which provided the basis of a model
of system for negative control of
protein synthesis ( Jacob &Monad
• In Pseudomonas putida ,
tryptophan synthetase is
induced by
indoleglycerophosphate and the
entire tryptophan branch is
induced by chorimate in B.
subtitles .
• Another induction system
involving positive control is
galactose utilization in
Saccharomyces cerevisiase .

32. COMPARTMENTALIZATION OF ENZYMES
• Enzyme compartmentalization is
efficiently carried out by
biomembrane or biological
membrane that permits to
confine specific functions given
by enzymes in a precise space.
The enzyme can be localized
inside the delimited
compartments or on the
membrane surface .
• In eukaryotic cells the
corresponding delimited spaces
Some of the organelles are
endoplasmic reticulum, Golgi
apparatus , nucleus ,
mitochondria , lysosomes ,
endosomes and peroxisomes.
Starting from nature simulation in
biotechnology process, enzyme
compartmentalization can be
obtained by immobilizing proteins
in supports or delimiting them by
support compartments .

33. DIFFERENTIAL ACTIVITY OF ISOENZYME
• Isoenzyme ( also called isozymes )
are alternative forms of the same
enzyme activity that exist in different
proportion in different tissue .
• Isoenzyme differ in amino acids
composition and sequence and
multimeric quarternary structure ,
mostly,but not always, they have
similar ( conserved ) structures .
• For example – Lactate
dehydrogenase- 5(LDH-5 ) , an
isoenzyme composed of 4 M
polypeptide chains , catalyzes the
conversion of pyruvate to lactate with
an unparalleled efficiency ( anaerobic
oxidation) , but this function of LDH
gradually fades as the number of H
over M chain increases .
• Figure :

34. REFERENCES
• https://www.karger.com/Article/Abstract/74430
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecular-biology/enzyme-induction
• https://www.sciencedirect.com/topics/neuroscience/isoenzyme
• https://www.thermofisher.com/in/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/overview-protein-protein-interaction-
analysis.html
• https://www.easybiologyclass.com/enzyme-regulation-mechanisms-
molecular-methods-regulate-enzyme-enzymatic-acitvity/
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecular-biology/phosphofructokinase-1
• https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology
_(Kaiser)/Unit_7%3A_Microbial_Genetics_and_Microbial_Metabolism/19%3A_