biochemical engineering lecture

8/12/2019 Biochemical Engineering Lecture

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Biochemical Engineering



BASICMICROBIOLOGY

http://www.microscopy-uk.org.uk/mag/wimsmall/extra/rotif4.html




http://www.microscopy-uk.org.uk/mag/artnov99/rotih.html



Protist Kingdom

Bacteria Blue green

algae

Fungi Algae Protozoa

Molds YeastEubacteria Archaeobacteria

Prokaryotes Eucaryotes

CLASSES OF ORGANISMS



PROKARYOTIC CELLS

They do not have a nucleus

They have no membrane-boundorganelles

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 membraneperforated with pores that allow transport ofmaterials back and forth to the cyotplasm.

The nucleus is the site of DNA replication andRNA synthesis (transcription). It is the site ofthe control of gene expression.



ROUGH ENDOPLASMIC RECTICULUM

It is rough because imbedded in the membrane areribosomes.

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 themembranes of the cell.

Ribosomes in the rough ER synthesize protein thatare then converted to glycoprotein and packaged in

transport vesicles for secretion.



SMOOTH ENDOPLASMIC RECTICULUM

The smooth ER is the site for the synthesis oflipids, 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 sexhormones.



The smooth ER of the liver has several additionalfunctions. Enzymes in the smooth ER regulate the

release of sugar into the bloodstream while otherenzymes break down toxic chemicals. As the liver isexposed to additional doses of a drug the liverincreases the amount of smooth ER to handle it. It

then takes more drug to get past the detoxifiyingability of the liver. We become more tolerant of thedrug.

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 seriesof folded membranes.

It functions in processing enzymes and other

products of the ER to a finished product.

It is the source of the production oflysosomes



MITOCHONDRIA

These organelles are the sites of respirationand convert the chemical energy of sugarsand other organic compounds into the high-energy phosphate bonds of an ATP molecule.

These are also bound by a doublemembrane. The inner membrane is thefolded (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 thesedigestive enzymes to the interior of thevacuole to digest the material inside thevacuole.



VACUOLES

These are membrane bound sacs that have

many different functions.

– The central vacuole of a plant cell serves as alarge lysosome.

– It may also function in absorbing water.

– The central vacuoles of flower petal cells may holdthe 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 wrappedin an extension of the plasma membrane.

It is sufficient to know that Energy is required to movethe cilia or flagella in a whip-like motion to propel thecell.



PROKARYOTIC CELLS EUCARYOTIC CELLS

1 – 5 m in length 10 – 30 m in length

Genetic material is a nucleoid, a poorlydemarcated region of the cell that lacks aboundary membrane to separate it fromsurrounding cytoplasm.

Possess a nucleus, a region bounded by acomplex membranous structure callednuclear envelop.

Contain relatively small amount of DNA

Length of DNA ranges from 0.25mm to3mm.

Single chromosome consists of essentiallynaked DNA

Contain several orders of magnitude ofmore genetic information

Length of DNA is about 4.6mm (yeast)

Chromosome consists of fibers containingboth DNA and protein

Cytoplasm is essentially devoid ofmembranous structure

Cytoplasm is filled with a great diversity ofstructures

No condensation of chromosomes and no

spindle apparatus. The DNA is duplicatedand the two copies are simply separatedby the growth of an intervening cellmembrane

Divide by a complex process of mitosis in

which duplicated chromosomes condenseinto compact structures that are separatedby an elaborate microtubule-containingapparatus

Simple locomotion mechanism Possess complex loco motor mechanism



The Chemistry of Life



The elements of life:

Stars (with assistance from the Big bang) have formed83 stable chemical elements in the universe

~95% of the mass of all terrestrial organisms composedof 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 ofextra cellular matrix



Monosaccharide / Disaccharide

DISACCHARIDES



DISACCHARIDES

MALTOSE

Lactose = -D-galactose + glucose



What is Starch?

The term starch is used to describe a biopolymer systemcomprising predominantly of two polysaccharides - amylose

and amylopectin.



AmyloseThe 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 activegroup on one end

– Fatty acids

– Neutral fats

– Phospholipids (fatty acid derivatives found incell membranes)

Structure formation is analogous tosurfactant, block copolymer



Lipid Classessimple: FA‟s esterified with glycerol compound: same as simple, but with other compoundsalso attached

phospholipids: fats containing phosphoric acid andnitrogen (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 andcombined w/FA‟s (e.g., cholesterol)



Nutritional Uses of Lipids

We already know that lipids are concentratedsources 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 forprostaglandin 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



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.

E ti l F tt A id



Essential Fatty AcidsNeeded by body but cannot be synthesized so

external source required

LINOLEIC CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH

18:2 n-6LINOLENIC CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH

18:3 n-3

EICOSOPENTAENOIC ACID

CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COO

H

20:5 n-3

DOCOSOHEXAENOIC ACID 22: 6 n-3



PROTEINS



Proteins

• Proteins are polymers composed of amino acid

monomers

• Polypeptides is another term for amino acid polymers

• Proteins are characterized by a specific primary

structure – order of mers in the backbone and DP

• Control of primary structure leads to control of 3Dstructure



Proteins

The control of protein structure buildsinformation into the molecule that translatesinto 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 andhydroxy proline is often present is the basicstuctural 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 chainconformations – 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 ofthree 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 offour pairs of the nitrogenous bases

– Either AT, TA, GC or CG



DNA Letters, Genes

The rungs of the double helix are like the mapon the floor. They spell out which amino acidshould 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 formedof three rungs of the double helix A set of three rungs is called a “gene”



DNA and amino acids

Each gene (three rungs) matches upchemically to one of the 20 aminoacids 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 thesequences of sets of three rungs

• They the amino acid monomers join together



The Nucleic Acid

Complex structures used tomaintain genetic information

DNA – deoxyribonucleic acidserves as the “Master Copy” formost information in the cell.

RNA – Ribonucleic acid acts totransfer information from DNA tothe rest of the cell.



The RNA Phosphoric acid

Ribose ( a pentose)

Organic (nitrogeneous) bases:

– Purines: Adenine & guanine

– Pyrimidines: Cytosine and Uracil



Th DNA



The DNA

DNA is a polymer known as

polynucleotide

• Each nucleotide consist of

-5 carbon sugar

-Nitrogen containing baseattached to the sugar

• There are 4 nucleotides

- Adenine- Guanine

- Thymine

- Cytosine



Translation of RNA to Protein



Translation of RNA to Protein

I iti ti

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Initiation



Elongation



Termination

U C

A G



U UUU = Phe

UUC = Phe

UUA = Leu

UUG = Leu

UCU = Ser

UCC = Ser

UCA = Ser

UCG = Ser

UAU = Tyr

UAC = Tyr

UAA = Stop

UAG = Stop

UGU = Cys

UGC = Cys

UGA = Stop

UGG = Trp U

C

A

G

C CUU = Leu

CUC = Leu

CUA = Leu

CUG = Leu

CCU = Pro

CCC = Pro

CCA = Pro

CCG = Pro

CAU = His

CAC = His

CAA = Gln

CAG = Gln

CGU = Arg

CGC = Arg

CGA = Arg

CGG = Arg U

C

A

G

A AUU = Ile

AUC = Ile

AUA = Ile

AUG = Met

ACU = Thr

ACC = Thr

ACA = Thr

ACG = Thr

AAU = Asn

AAC = Asn

AAA = Lys

AAG = Lys

AGU = Ser

AGC = Ser

AGA = Arg

AGG = Arg U

C

A

G

G GUU = ValCUC = Val

GUA = Val

GUG = Val GCU = AlaGCC = Ala

GCA = Ala

GCG = Ala GAU = AspGAC = Asp

GAA = Glu

GAG = Glu GGU = GlyGCG = Gly

GGA = Gly

GGG = Gly U

C

A

G

AUG = start codon

UAA, UAG, and UGA = stop (nonsense) codons

Amino Acids



Amino Acids

Phe = phenylalanine

Leu = leucine

Ile = isoleucine

Met = methionine

Val = valine

Ser = serine

Pro = proline

Thr = threonine

Ala = alanine

Tyr = tyrosine

His = histidineGln = glutamine

Asn = asparagine

Lys = lysine

Asp = aspartic acid

Glu = glutamic acid

Cys = cysteine

Trp = tryptophanArg = arginine

Gly = glycine

S t M t ti



Spontaneous Mutation

•

Substitution of a nucleotide (point mutations)




•Deletion or addition of a nucleotide


Results of Spontaneous Mutation



Results of Spontaneous Mutation

Missense mutation This is usually seenwith 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 ornonsense 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

E Bi l i l



– Enzymes as BiologicalCatalysts

Increase reaction ratesby over 1,000,000-fold

Two fundamentalproperties

– Increase the reaction ratewith no alteration of theenzyme

– Increase the reaction ratewithout altering the

equilibrium Reduce the activation

energy



– Enzymes as Biological Catalysts

The substrate binds to a specific region called theactive site



– Enzymes as Biological

Catalysts Two popular models

provide an aid tounderstanding the

mechanisms of enzymeaction:

– Lock-and-key

– Induced fit

The M ichaelis Menten Equation



The M ichaelis-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 equilibriumwith 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



V

[S]

(Vmax)

E + S ES E + Pk 1 k 2



E + S ES E + Pk -1 k -2

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]

E + S ES E + Pk 1 k 2



E + S ES E + Pk -1

[Et] [S][ES] = -------------------------

[S] + (K2+ K-1) / K1

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

(as the Michaelis-Menten constant)

[Et] [S]

[ES] = -----------------

[S] + km

E + S ES E + Pk 1 k 2



E + S ES E + Pk -1

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)

km + [S]

E + S ES E + Pk 1 k 2



E + S ES E + Pk -1

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 K



Understanding K m

The “kinetic activator constant”

K m is a constant

K m is constant derived from rate constants

K m is, under true Michaelis-Meten conditions,estimate of the dissociation constant of E from S

Small K m means tight binding; high K m means weak

binding



Understanding V max

The theoretical maximal velocityVmax 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 M ichaelis-



The dual nature of the M ichaelis

Menten equation

Combination of 0-order and 1 st -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 SThe Michaelis-Menten equation diescribes a

rectangular hyperbolic dependence of v on S!

Th b



The turnover number

A measure of catalytic activity

k cat, the turnover number, is the number of

substrate molecules converted to product per

enzyme molecule per unit of time, when E issaturated with substrate

If the M-M model fits, k 2 = k cat = Vmax/Et

Values of k cat

range from less than 1/sec to many

millions per sec

Vmax [S]



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

( This is the Lineweaver-Burk equation)

Lineweaver-Burk Plot: 1/V0 against 1/[S]



0 g [ ]



Enzyme I nhibitors



Enzyme I nhibitors

Competitive vs. Noncompetitive

SvCompetitive Inhibition



I

M K I S K

S v v

max

Competitive Inhibition



Non-Competitive Inhibition

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