Biochemical Engineering

BASIC
MICROBIOLOGY
 CLASSES OF ORGANISMS

Protist Kingdom

Prokaryotes Eucaryotes

Bacteria Blue green Fungi Algae Protozoa

algae

Eubacteria Archaeobacteria Molds Yeast

 PROKARYOTIC CELLS
 They do not have a nucleus
 They have no membrane-bound
organelles
 The parts are
– Cell wall
– Plasma membrane
– Ribosomes
– Flagella
– Pili
The Bacterial Cell
 EUKARYOTIC CELLS
 Plant Cell
 Animal Cell
 Parts
– Nucleus
– Plasma membrane
– Organelles
 Endoplasmic recticulum
– Rough
– Smooth
 Golgi Complex
 Mitochondrion
 Lysosome
 Chloroplast
An Animal Cell
A Plant Cell
 THE NUCLEUS
 It houses the chromatin, which is a mass of
DNA and protein.
 During cell division the chromatin coils up into
recognizable chromosomes.
 The nuclear envelope is a double membrane
perforated with pores that allow transport of
materials back and forth to the cyotplasm.
 The nucleus is the site of DNA replication and
RNA synthesis (transcription). It is the site of
the control of gene expression.
 ROUGH ENDOPLASMIC RECTICULUM
 It is rough because imbedded in the membrane are
ribosomes.
 It is the site of the synthesis of secretory proteins.
 It is the site for the synthesis of membrane. Enzymes
synthesize phospholipid that forms all the
membranes of the cell.
 Ribosomes in the rough ER synthesize protein that
are then converted to glycoprotein and packaged in
transport vesicles for secretion.
SMOOTH ENDOPLASMIC RECTICULUM

 The smooth ER is the site for the synthesis of

lipids, phospholipids, and steroids.
 The production of steriod hormones is tissue
specific.
– For example, it is the smooth ER of the cells of the
ovaries and testes that synthesize the sex
hormones.
 The smooth ER of the liver has several additional
functions. Enzymes in the smooth ER regulate the
release of sugar into the bloodstream while other
enzymes break down toxic chemicals. As the liver is
exposed to additional doses of a drug the liver
increases the amount of smooth ER to handle it. It
then takes more drug to get past the detoxifiying
ability of the liver. We become more tolerant of the
drug.

 Finally the smooth ER functions to store calcium ions.

Ca+ ions are required for muscle contraction.
 THE GOLGI COMPLEX
 The Golgi apparatus, like the ER, is a series of
folded membranes.

 It functions in processing enzymes and other

products of the ER to a finished product.

 It is the source of the production of

lysosomes
 MITOCHONDRIA
 These organelles are the sites of respiration
and convert the chemical energy of sugars
and other organic compounds into the high-
energy phosphate bonds of an ATP molecule.

 These are also bound by a double

membrane. The inner membrane is the
folded (the folds are called cristae) and is the
site of the electron transport system.
 LYSOSOMES
 These are membrane bound vesicles that
harbor digestive enzymes.

 The membrane of a lysosome will fuse with

the membrane of vacuoles and releases these
digestive enzymes to the interior of the
vacuole to digest the material inside the
vacuole.
 VACUOLES
 These are membrane bound sacs that have
many different functions.

– The central vacuole of a plant cell serves as a large

lysosome.

– It may also function in absorbing water.

– The central vacuoles of flower petal cells may hold

the pigments that give the flower its color.

– The contractile vacuoles of protists collect and

excrete water.
Lysosome Formation and Function
 CILIA AND FLAGELLA
 These are found on cells, such as protists, that are
motile.

 Cilia are short and numerous.

 Flagella are longer and less numerous appendages

 These are composed of a core of microtubules wrapped

in an extension of the plasma membrane.

 It is sufficient to know that Energy is required to move

the cilia or flagella in a whip-like motion to propel the
cell.
 PROKARYOTIC CELLS
1 – 5 m in length
Genetic material is a nucleoid, a poorly
demarcated region of the cell that lacks a
boundary membrane to separate it from
surrounding cytoplasm.
Contain relatively small amount of DNA
Length of DNA ranges from 0.25mm to
3mm.
Single chromosome consists of essentially Chromosome consists of fibers containing
naked DNA
Cytoplasm is essentially devoid of
membranous structure
No condensation of chromosomes and no
spindle apparatus. The DNA is duplicated
and the two copies are simply separated
by the growth of an intervening cell
membrane
Simple locomotion mechanism
EUCARYOTIC CELLS
10 – 30 m in length
Possess a nucleus, a region bounded by a
complex membranous structure called
nuclear envelop.
Contain several orders of magnitude of
more genetic information
Length of DNA is about 4.6mm (yeast)
both DNA and protein
Cytoplasm is filled with a great diversity of
structures
Divide by a complex process of mitosis in
which duplicated chromosomes condense
into compact structures that are separated
by an elaborate microtubule-containing
apparatus
Possess complex loco motor mechanism
The Chemistry of Life
 The elements of life:
 Stars (with assistance from the Big bang) have formed
83 stable chemical elements in the universe

 ~95% of the mass of all terrestrial organisms composed

of just 4 of them
– Hydrogen (61% in humans)
– Oxygen (26% in humans)
– Carbon (10.5% in humans)
– Nitrogen (2.4% in humans)
CARBOHYDRATES
 Polysaccharides
 Polymers composed of sugars /
saccharides
 Uses include energy source, component of
extra cellular matrix
Monosaccharide / Disaccharide
DISACCHARIDES

MALTOSE

Lactose = -D-galactose + glucose

 What is Starch?
The term starch is used to describe a biopolymer system
comprising predominantly of two polysaccharides - amylose
and amylopectin.
Amylose
The smaller of the two polysaccharides which make up starch, amylose is a linear
molecule comprising of (1-4) linked alpha-D-glucopyranosyl units.

Figure 2 : Amylose molecule

Amylopectin
The larger of the two components, amylopectin is highly branched with a much
greater molecular weight. This structure contains alpha-D-glucopyranosyl units
linked mainly by (1-4) linkages (as amylose) but with a greater proportion of (1-6)
linkages, which gives a large highly branched structure.

Amylopectin has been found to form the basis of the structure of starch granules.
This is because the short branched (1-4) chains are able to form helical structures
which crystallise.
 Cellulose : the major structural component of woody
plants and natural fibers such as cotton, wood, and cork, is a
ß-D-glucose polymer found in vegetable matter.

The ß-glycoside linkages in cellulose give the glucose rings a different relative
orientation than is found in starch. Although this difference may seem minor, it
has very important consequences : human being are not able to digest them !
LIPIDS
 Lipids and Phospholipids
 Long hydrocarbon chains with active
group on one end
– Fatty acids
– Neutral fats
– Phospholipids (fatty acid derivatives found in
cell membranes)
 Structure formation is analogous to
surfactant, block copolymer
 Lipid Classes
simple: FA’s esterified with glycerol
compound: same as simple, but with other compounds
also attached
phospholipids: fats containing phosphoric acid and
nitrogen (lecithin)
glycolipids: FA’s compounded with CHO, but no N
derived lipids: substances from the above derived by
hydrolysis
sterols:large molecular wt. alcohols found in nature and
combined w/FA’s (e.g., cholesterol)
 Nutritional Uses of Lipids

We already know that lipids are concentrated

sources of energy (9.45 kcal/g)
other functions include:

1) provide means whereby fat-soluble nutrients

(e.g., sterols, vitamins) can be absorbed by the body
2) structural element of cell, subcellular components
3) components of hormones and precursors for
prostaglandin synthesis
Omega fatty acids

Polyunsaturated fatty acids like DHA and EPA are added to

many foods to due to their nutritive value. They are present
naturally to the highest levels in fish oils.
Triglycerides
 A FAT MOLECULE = GLYCEROL + FATTY ACID

The three fatty acids in a single fat molecule may be all alike (as
shown here for tristearin) or they may be different. They may
contain as few as 4 carbon atoms or as many as 24.
 Essential Fatty Acids
Needed by body but cannot be synthesized so
external source required

LINOLEIC CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH
18:2 n-6
LINOLENIC CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH
18:3 n-3
EICOSOPENTAENOIC ACID
CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COOH
20:5 n-3
DOCOSOHEXAENOIC ACID 22: 6 n-3
PROTEINS
 Proteins
 The control of protein structure builds information
into the molecule that translates into function
 Proteins are the most common biological
macromolecules in the extra cellular matrix
 Perform structural and functional tasks
– Collagen (triple helix – gly-X-Y) where proline and hydroxy
proline is often present is the basic stuctural protein
– Enzymes perform specific catalytic tasks
– Adhesive proteins are bind cells to substrates –
fibronectin, integrin, etc.
– Provide signal transduction between cells and ECM
Peptide Synthesis
 Protein Structure Hierarchy
 Secondary structure refers to local chain
conformations – four types are known:
  helix – regular helix
  sheet – extended zig-zag
  turn – puts fold into  sheet
– Globular or random coil
 Tertiary structure refers to secondary structure
stabilized by H bonds – defines protein folding
NUCLEIC ACIDS
 DNA Chemistry
 DNA is a complex molecule which is built of
three basic types of monomers:
– 1. Sugar (deoxyribose)
– 2. A phosphate PO4
– 3. One of four “nitrogenous bases”
 Adenine (A)
 Guanine (G)
 Cytosine (C)
 Thymine (T)
– These four monomers are collectively called
“nucleotides”
The DNA Nitrogenous Bases:
Differences between
DNA and RNA
 DNA construction
 The double helix:
– Resembles a twisted ladder
 The “rails” of the DNA ladder are made of the
sugar and phosphate

 The “rungs” of the ladder are composed of one of

four pairs of the nitrogenous bases
– Either AT, TA, GC or CG
 DNA Letters, Genes
 The rungs of the double helix are like the map
on the floor. They spell out which amino acid
should line up where
– Each rung can have one of four possible “letters”
 AT
 TA
 GC
 CG
– Each slot where an amino acid will line up is formed
of three rungs of the double helix
 A set of three rungs is called a “gene”
 DNA and amino acids
 Each gene (three rungs) matches up
chemically to one of the 20 amino
acids used by life
• Each gene ‘spells’ the name of an amino acid!
• The amino acids line up along the double helix
according to the map spelled out by the
sequences of sets of three rungs
• They the amino acid monomers join together
 The Nucleic Acid

Complex structures used to

maintain genetic information
 DNA – deoxyribonucleic acid
serves as the “Master Copy” for
most information in the cell.
 RNA – Ribonucleic acid acts to
transfer information from DNA to
the rest of the cell.
 The RNA
 Phosphoric acid
 Ribose ( a pentose)
 Organic (nitrogeneous) bases:
– Purines: Adenine & guanine
– Pyrimidines: Cytosine and Uracil
 The DNA
DNA is a polymer known as
polynucleotide

• Each nucleotide consist of

-5 carbon sugar
-Nitrogen containing base
attached to the sugar

• There are 4 nucleotides

- Adenine
- Guanine
- Thymine
- Cytosine
Translation of RNA to Protein
Initiation
Elongation
Termination
  U C A G  

UUU = Phe UCU = Ser UAU = Tyr UGU = Cys

UUC = Phe UCC = Ser UAC = Tyr UGC = Cys U
UUA = Leu UCA = Ser UAA = Stop UGA = Stop C
U
UUG = Leu UCG = Ser UAG = Stop UGG = Trp A
G

CUU = Leu CCU = Pro CAU = His CGU = Arg

CUC = Leu CCC = Pro CAC = His CGC = Arg U
CUA = Leu CCA = Pro CAA = Gln CGA = Arg C
C
CUG = Leu CCG = Pro CAG = Gln CGG = Arg A
G

AUU = Ile ACU = Thr AAU = Asn AGU = Ser

AUC = Ile ACC = Thr AAC = Asn AGC = Ser U
AUA = Ile ACA = Thr AAA = Lys AGA = Arg C
A
AUG = Met ACG = Thr AAG = Lys AGG = Arg A
G

GUU = Val GCU = Ala GAU = Asp GGU = Gly

CUC = Val GCC = Ala GAC = Asp U
GCG = Gly
GUA = Val GCA = Ala GAA = Glu C
G GGA = Gly A
GUG = Val GCG = Ala GAG = Glu
GGG = Gly G

AUG = start codon

UAA, UAG, and UGA = stop (nonsense) codons
 Amino Acids
Phe = phenylalanine His = histidine
Leu = leucine Gln = glutamine
Ile = isoleucine Asn = asparagine
Met = methionine Lys = lysine
Val = valine Asp = aspartic acid

Ser = serine Glu = glutamic acid

Pro = proline Cys = cysteine
Thr = threonine Trp = tryptophan
Ala = alanine Arg = arginine
Tyr = tyrosine Gly = glycine
 Spontaneous Mutation
•Substitution of a nucleotide (point mutations)
 Spontaneous Mutation

•Deletion or addition of a nucleotide

Results of Spontaneous Mutation
 Missense mutation This is usually seen
with a single substitution mutation and
results in one wrong codon and one
wrong amino acid
 Nonsense mutation - If the change in the
deoxyribonucleotide base sequence
results in transcription of a stop or
nonsense codon, the protein would be
terminated at that point in the message
 Sense mutation - This is sometimes
seen with a single substitution
mutation when the change in the
DNA base sequence results in a new
codon still coding for the same
amino acid.
 Frameshift Mutation - This is seen when a
number of DNA nucleotides not divisible
by three is added or deleted.
ENZYME KINETICS AND
APPLICATIONS
 Enzymes

• Enzymes endow cells with the remarkable capacity

to exert kinetic control over thermodynamic
potentiality

• Enzymes are the agents of metabolic function

ENZYMES ARE NAMED BY WHAT THEY DO RATHER THAN WHAT THEY
ARE. NAME ENDS WITH …-ASE. EG. AMYLASE
– Enzymes as Biological
Catalysts
 Increase reaction rates
by over 1,000,000-fold
 Two fundamental
properties
– Increase the reaction rate
with no alteration of the
enzyme
– Increase the reaction rate
without altering the
equilibrium
 Reduce the activation
energy
– Enzymes as Biological Catalysts
 The substrate binds to a specific region called the
active site
– Enzymes as Biological
Catalysts
 Two popular models
provide an aid to
understanding the
mechanisms of enzyme
action:
– Lock-and-key
– Induced fit
 The Michaelis-Menten Equation

Louis Michaelis and Maude Menten’s theory

It assumes the formation of an enzyme-substrate
complex
It assumes that the ES complex is in rapid equilibrium
with free enzyme
Breakdown of ES to form products is assumed to be
slower than 1) formation of ES and 2) breakdown of ES
to re-form E and S
Plot initial velocity against substrate concentration

(Vmax)

[S]
 k1 k2
E + S k-1
ES k-2
E + P

The following assumptions allow Michaelis-Menten model to

explain V vs S kinetics

1. Enzyme and substrate combine to form ES complex

2. Assume reverse rxn, k-2, is negligible

3. Assume [ES] is constant, steady state assumption: d[ES]/dt = 0

4. [E] <<<[S]
 k1 k2
E + S k-1
ES E + P
[Et] [S]
[ES] = -------------------------
[S] + (K2+ K-1) / K1

Define Km as (K2+ K-1) / K1

(as the Michaelis-Menten constant)

[Et] [S]
[ES] = -----------------
 k1 k2
E + S k-1
ES E + P
V0 = K2 [ES]

K2 [Et] [S]
= ------------------
[S] + km

Maximum velocity (Vmax) occurs when [ES] = [Et]

Thus, Vmax = K2 [Et]

Vmax [S]
V0 = ------------------ (Michaelis-Menten equation)
 k1 k2
E + S k-1
ES E + P
Vmax [S]
V0 = ------------------
km + [S]

When V0 = 1/2 Vmax

Vmax Vmax [S]

--------------- = ------------------
2 km + [S]

Solve for km

Km = [S] when V0 = 1/2 Vmax

Let's find Vmax & Km on the graph

Vmax = v at highest [S]

Km = [S] at 1/2 Vmax

 Understanding Km

The “kinetic activator constant”

Km is a constant
Km is constant derived from rate constants
Km is, under true Michaelis-Meten conditions,
estimate of the dissociation constant of E from S
Small Km means tight binding; high Km means weak
binding
 Understanding Vmax

The theoretical maximal velocity

Vmax is a constant
Vmax is the theoretical maximal rate of the reaction –
but it is NEVER achieved in reality
To reach Vmax would require that all enzyme
molecules are tightly bound with subtrate
Vmax is asymptotically approached as substate is
increased
 The dual nature of the Michaelis-
Menten equation
Combination of 0-order and 1st-order kinetics

When S is low, the equation for rate is 1st-order in S

When S is high, the equation for rate is 0-order in S
The Michaelis-Menten equation diescribes a
rectangular hyperbolic dependence of v on S!
 The turnover number

A measure of catalytic activity

kcat, the turnover number, is the number of substrate

molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate
If the M-M model fits, k2 = kcat = Vmax/Et
Values of kcat range from less than 1/sec to many
millions per sec
 Vmax [S]
V0 = ------------------
km + [S]

1 km + [S]
--------- = ------------------
V0 Vmax [S]

1 km [S]
---------- = ------------ + ------------
V0 Vmax [S] Vmax [S]

1 km 1 1
---------- = ------ ------ + ------
V0 Vmax [S] Vmax
Lineweaver-Burk Plot: 1/V0 against 1/[S]
Not all enzymes obey Michaelis- Menten kinetics:
allosteric, regulatory enzymes
 Enzyme Inhibitors

Competitive vs. Noncompetitive

Competitive Inhibition v max S
v
KM  S  I
KI
Non-Competitive Inhibition