Chapter 2 Nutrition and Culture

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Chapter 2 Journey to Microbial

World



Chapter 4 Nutrition and Culture

of Microorganisms



Different chemical reactions and organizemany different molecules into specific

structures is known as metabolism

Catabolism breaks molecular structures down,releasing energy in the process, and

anabolism uses energy to build larger

molecules from smaller ones.

Metabolic reactions are either catabolic,which means energy releasing, or anabolic,

which means energy requiring.



All microbial nutrients are compoundsconstructed from the chemical elements.

However, just a handful of elements

dominate living systems and areessential: hydrogen (H), oxygen (O),

carbon (C), nitrogen (N), phosphorus

(P), sulfur (S), and selenium (Se). Inaddition to these, at least 50 other

elements, although not required, are

metabolized in some way by

microorganisms



Besides water, which makes up 70 –80%of the wet weight of a microbial cell (a

single cell of Escherichia coli weighs just

g), cells consist primarily ofmacromolecules—proteins, nucleic

acids, lipids, and polysaccharides.



All cells require carbon, and mostprokaryotes require organic (carbon-

containing) compounds as their source

of carbon. Heterotrophic bacteria assimilate

organic compounds and use them to

make new cell material. Autotrophic microorganisms build their

cellular structures from carbon dioxide

(CO2) with energy obtained from light or



Other Macronutrients: P, S, K,Mg,

Ca, Na

• In addition to C, N, O, and H, many otherelements are needed by cells, but in smaller

amounts .

• Phosphorus is a key element in nucleic acidsand phospholipids and is typically supplied to

a cell as phosphate (PO4)

• Sulfur is present in the amino acids cysteine

and methionine and also in several vitamins,including thiamine, biotin, and lipoic acid.

Sulfur can be supplied to cells in several

forms, including sulfide (HS2) and sulfate

(SO4)



Potassium (K) is required for the activityof several enzymes, whereasmagnesium

(Mg) functions to stabilize ribosomes,membranes, and nucleic acids and isalso required for the activity of manyenzymes.

Calcium (Ca) is not required by all cellsbut can play a role in helping to stabilizemicrobial cell walls, and it plays a keyrole in the heat stability of endospores.



Sodium (Na) is required by some, but not all,microorganisms, and its requirement is

typically a reflection of the habitat. For

example,seawater contains relatively high

levels of Na, and marine

microorganisms typically require Na for

growth.

By contrast, freshwater species are usuallyable to grow in the absence of Na.

K, Mg, Ca, and Na are all supplied to cells as

salts, typically as chloride or sulfate salts.



Micronutrients: Iron and Other

Trace Metals

Microorganisms require several metalsfor growth

Chief among these is iron (Fe), which

plays a major role in cellular respiration.Iron is a key component of cytochromes

and of iron –sulfur proteins involved in

electron transport reactions . Under anoxic conditions, iron is

generally in the ferrous form and

soluble. However, under oxic conditions,



Defined media are prepared by adding preciseamounts of highly purified inorganic or organic

chemicals to distilled water.

Therefore, the exact composition of a defined

medium (in both a qualitative and quantitativesense) is known.

Major importance in any culture medium is the

carbon source because all cells need large

amounts of carbon to make new cell material



For culturing many microorganisms, knowledge ofthe exact

composition of a medium is not essential. In

these instances

complex media may suffice and may even be

advantageous.

Complex media employ digests of microbial,

animal or plant products, such as casein (milkprotein), beef (beef extract), soybeans (tryptic soy

broth), yeast cells (yeast extract), or any of a

number of other highly nutritious yet impure

substances.



An enriched medium, often used for theculture of otherwise difficult-to-grow

nutritionally demanding (fastidious)

microorganisms, starts with a complexbase and is embellished with additional

nutrients such as serum, blood, or other

highly nutritious substances.



A selective medium contains compounds thatinhibit the growth of some microorganisms but

not others. For example, media are available

for the selective isolation

of pathogenic strains of E. coli from food

products, such as ground beef, that could be

contaminated with this organism.



Differential medium is one in which anindicator, typically a reactive dye, is

added that reveals whether a particular

chemical reaction has occurred duringgrowth.



Solid and Liquid Culture Media

Liquid culture media are sometimessolidified by the addition of a gelling

agent.

Solid media immobilize cells,

allowing them to grow and form

visible, isolated masses called

colonies.



Solid and Liquid Culture Media

Microbial colonies are of various shapesand sizes depending on the organism,

the culture conditions, the nutrient

supply, and several other physiologicalparameters, and can contain several

billion individual cells.

Some microorganisms producepigments that cause the colony to be

colored. Colonies permit the

microbiologist to visualize the

com osition and resum tive



Solid media are prepared in the same way asliquid media

except that before sterilization, agar, a gelling

agent, is added to the medium, typically at a

concentration of 1 –2%.

The agar melts during the sterilization process,

and the molten medium is then poured into sterile

glass or plastic plates and allowed to solidify

before use.



OBJECTIVES:

By the end of the lesson, student should be ableto :

1. define terms enzyme;

2. understand the Lock & Key model andInduced-fit model;

3. identify the groups of enzymes; and

4. understand some factors affecting the

enzyme activities.



Enzymes are proteins that catalyze (i.e. accelerate) and control the rates of chemical

reactions.

In enzymatic reactions, the molecules at the

beginning of the process are called substrates,and the enzyme converts them into different

molecules, the products.

Almost all processes in a biological cell needenzymes in order to occur at significant rates.



Since enzymes are extremely selective for theirsubstrates and speed up only a few reactions

from among many possibilities, the set of

enzymes made in a cell determines which

metabolic pathways occur in that cell.

ENZYME AS BIOLOGICAL



ENZYME AS BIOLOGICAL

CATALYSTS:

Enzymes are biological catalysts produced byliving cells.

Enzymes lower the amount of activation energyneeded.

They speed up the rate of biochemical reactionsin the cell but remain unchanged at the end of thereactions.

Most enzymes are globular protein molecules.



The chemicals which an enzyme acts on is called its

substrate. The enzyme combines with its substrate to form an

enzyme-substrate complex.

The complex than breaks up into product and enzyme.

A metabolic pathway is a number of reactions catalysedby sequence of enzymes.



MECHANISM ACTION:

There are 2 main hypotheses explaining of

enzyme action.



Each enzyme is specific for one and ONLY one

substrate (one lock - one key)

active site: part of the enzyme that fits with the

substrate

Note that the active site has a specific fit for this

particular substrate and no other.

This theory has some weaknesses, but it explains

many basic things about enzyme function.



Substrate: The starting molecules for a chemicalreaction are called the substrates.

Enzyme substrate complex: The enzymesubstrate complex is transitional step when thesubstrates of a chemical reaction are bound tothe enzyme.

Active site: The area on the enzyme where thesubstrate or substrates attach to is called theactive site.

Enzymes are usually very large proteins and theactive site is just a small region of the enzymemolecule.



The induced-fit theory assumes that the substrate

plays a role in determining the final shape of the

enzyme and that the enzyme is partially flexible.

This explains why certain compounds can bind tothe enzyme but do not react because the enzyme

has been distorted too much.

Other molecules may be too small to induce the

proper alignment and therefore cannot react.

Only the proper substrate is capable of inducing the

proper alignment of the active site.



http://en.wikipedia.org/wiki/Image:Induced_fit_diagram.svg



In the graphic, the substrate is represented by themagenta molecule, the enzyme protein is

represented by the green and cyan colors.

The cyan colored protein is used to more sharply

define the active site. The protein chains are flexible and fit around the

substrate.



Both are used by enzymes and have been

evolutionarily chosen to minimize the ΔG of thereaction.

Enzymes which are saturated, ie. have a high affinitysubstrate binding, require differential binding to

reduce the ΔG, whereas largely substrate unboundenzymes may use either differential or uniformbinding.



How do enzymes work?

substrate: molecules upon which an enzymeacts. The enzyme is shaped so that it can only

lock up with a specific substrate molecule.

enzyme

substrate -------------> product



The diagram shows time on the horizontal axis and

the amount of energy in the chemicals involved in a

chemical reaction on the vertical axis.

The point if this diagram again is that without the

enzyme, much more activation energy is required toget a chemical reaction to take place.



Factors Influencing Enzyme Activity

pH: the optimum (best) in most living things isclose to 7 (neutral).

High or low pH levels usually slow enzyme

activity



Temperature: strongly influences enzyme

activity optimum (best) temperature for maximumenzyme function is usually about 35-40 C.

Reactions proceed slowly below optimal

temperatures.

Above 45 C. most enzymes are denatured (change in their shape so the enzyme active site

no longer fits with the substrate and the enzyme

can't function)



METABOLISM

Metabolism is the sum of all biochemicalreactions occurring in living cells.

These reactions can be divided into two main

groups:

1) ANABOLISM 2) CATABOLISM



Involves the synthesis

of complex molecules

from simpler moleculeswhich requires energy

input.

Involves the

breakdown of complex

molecules into simplermolecules involving

hydrolysis or oxidation

and the release of

energy.



Energy releasing processes, ones that "generate"energy, are termed exergonic reactions.

Reactions that require energy to initiate thereaction are known as endergonic reactions.

All natural processes tend to proceed in such adirection that the disorder or randomness of theuniverse increases



In an exergonic reaction the change is freeenergy is represented by a negative number (-

G), indicating free energy is released during

the reaction.



This kind of reaction is not termed a

spontaneous reaction. In order to go from the

initial state to the final state a considerable

amount of energy must be imparted to the

system.

These kinds of reactions are associated with a

positive number (+G).



Th d V th b f ti



The speed V means the number of reactions per

second that are catalyzed by an enzyme.

With increasing substrate concentration [S], theenzyme is asymptotically approaching its

maximum speed V max, but never actually

reaching it.

Because of that, no [S] for V max can be given.

Instead, the characteristic value for the enzyme

is defined by the substrate concentration at its

half-maximum speed (V max /2 ). This KM value is also called Michaelis-Menten

constant.



V o =

V maxKM

Vo = Initial reaction velocity Vmax = Maximum velocity

Km = Michaelis constant

[S] = Substrate concentration



A non protein component of enzymes is called thecofactor.

If the cofactor is organic, then it is called a

coenzyme.

Coenzymes are relatively small moleculescompared to the protein part of the enzyme.

Many of the coenzymes are derived from vitamins.

The coenzymes make up a part of the active site,since without the coenzyme, the enzyme will not

function.



In the graphic on the left is the structure forthe coenzyme, NAD+, Nicotinamide

Adenine Dinucleotide.

Nicotinamide is from the niacin vitamin. The NAD+ coenzyme is involved with

many types of oxidation reactions where

alcohols are converted to ketones or

aldehydes.



Vitamin Coenzyme Function

niacin nicotinamide adeninedinucleotide (NAD+) oxidation orhydrogen transfer

riboflavinflavin adenine

dinucleotide (FAD)

oxidation or

hydrogen transfer

pantothenic

acidcoenzyme A (CoA) Acetyl group carrier

vitamin B-12 coenzyme B-12Methyl group

transfer

thiamin (B-1)thiaminpyrophosphate

(TPP)

Aldehyde group

transfer



Enzyme inhibitors are molecules that interact in someway with the enzyme to prevent it from working in the

normal manner.

There are a variety of types of inhibitors including:

nonspecific, irreversible, reversible - competitive and

noncompetitive.

Poisons and drugs are examples of enzyme inhibitors.



A nonspecific inhibition effects all enzymes in thesame way.

Non-specific methods of inhibition include any

physical or chemical changes which ultimately

denatures the protein portion of the enzyme andare therefore irreversible.



The inhibitor is "stuck" on the enzyme andprevents any substrate molecules fromreacting with the enzyme.

However, a competitive inhibition is usually

reversible if sufficient substrate molecules areavailable to ultimately displace the inhibitor.

Therefore, the amount of enzyme inhibitiondepends upon the inhibitor concentration,

substrate concentration, and the relativeaffinities of the inhibitor and substrate for theactive site.



There are approximately 3000 enzymes whichhave been characterised.

These are grouped into six main classesaccording to the type of reaction catalysed.

At present, only a limited number are used inenzyme electrodes or for other analyticalpurposes.

1 O id d t



1.Oxidoreductases

These enzymes catalyse oxidation and reductionreactions involving the transfer of hydrogen

atoms or electrons.

The following are of particular importance in the

design of enzyme electrodes. This group can be further divided into 4 main

classes.



catalyse hydrogen transfer from the substrate to

molecular oxygen producing hydrogen peroxide as a

by-product. An example of this is FAD dependent

glucose oxidase which catalyses the following reaction:

b-D-glucose + O2 = gluconolactone + H2O2

oxidases



dehydrogenases

catalyse hydrogen transfer from the substrate to anicotinamide adenine dinucleotide cofactor

(NAD+). An example of this is lactate

dehydrogenase which catalyses the following

reaction:

Lactate + NAD+ = Pyruvate + NADH + H+

peroxidases



p

catalyse oxidation of a substrate by hydrogen peroxide.

An example of this type of enzyme is horseradishperoxidase which catalyses the oxidation of a number of

different reducing substances (dyes, amines,

hydroquinones etc.) and the concomitant reduction of

hydrogen peroxide.

The reaction below illustrates the oxidation of neutral

ferrocene to ferricinium in the presence of hydrogen

peroxide:

2[Fe(Cp)2] + H2O2 + 2H+= 2[Fe(Cp)2]+ + 2 H2O



catalyse substrate oxidation by molecular oxygen.

The reduced product of the reaction in this case is

water and not hydrogen peroxide.

An example of this is the oxidation of lactate to acetatecatalysed by lactate-2-monooxygenase.

lactate + O2 = acetate + CO2 + H2O

oxygenases

2 T f



2.Transferases

These enzymes transfer C, N, P or S containinggroups (alkyl, acyl, aldehyde, amino, phosphate

or glucosyl) from one substrate to another.

Transaminases, transketolases, transaldolases

and transmethylases belong to this group.

3 H d l



3.Hydrolases

These enzymes catalyse cleavage reactions or thereverse fragment condensations.

According to the type of bond cleaved, a distinction ismade between peptidases, esterases, lipases,

glycosidases, phosphatases and so on. Examples of this class of enzyme include; cholesterol

esterase, alkaline phosphatase and glucoamylase.



5 I



5.Isomerases

These enzymes catalyse intramolecularrearrangements and are subdivided into;o racemases

o epimerases

o mutases

o c is -t rans -isomerases

An example of this class of enzyme is glucose

isomerase which catalyses the isomerisation of

glucose to fructose.

6 Li



6.Ligases

Ligases split C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or

oxidation.

The reaction is usually accompanied by the

consumption of a high energy compound suchas ATP and other nucleoside triphosphates.

An example of this type of enzyme is

pyruvate carboxylase which catalyses thefollowing reaction:

pyruvate + HCO3- + ATP = Oxaloacetate + ADP + Pi

IEC Classification of Enzymes

G N T f R ti C t l d



Group Name Type of Reaction Catalyzed

Oxidases or

Dehydrogenases

Oxidation-reduction

reactions

Transferases Transfer of functional

groups

Hydrolases Hydrolysis reactions

Lyases Addition to double bonds or

its reverse

Isomerases Isomerization reactions

Ligases or Synthetases Formation of bonds with

ATP cleavage



Enzymes do NOT change the equilibrium position of thereaction, just the speed at which equilibrium is attained.

Most are globular or soluble.

Stereospecific (can recognize certain isomers only) due tothe fact that amino acids of the active site are chiralthemselves.

Substrate/s bind in hydrophobic cleft (active site) betweenseveral domains where catalysis occurs: Van der Waals forces

Hydrophobic interactions Electrostatic interactions

Active site has structure that is complimentary in structure tothe structure of its substrate.



Most are proteins, some are RNA.

Biological catalysts.

E + S ES EP E + P Not changed by the reaction overall Much higher reaction rates than uncatalyzed reactions. Allow for biochemical reactions to occur under very mild

conditions (temperature, near-neutral pH, 1 atm pressure) High yield of products (few side reactions or by-products)

Very specific reactions (specific for its substrate or a family ofrelated substrates)

Often a regulated functions:

allosteric activation or inhibition covalent modification (phosphorylation changes) enzyme expression controlled or cleavage of proenzyme

controlled.



Describe what metabolism is? What is the difference between anabolism and

catabolism?

What is a substrate?

List 6 types of enzyme and state thecharacteristics each of them.

Chapter 2 Nutrition and Culture

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