Final summary
Lesson 1 : Carbohydrate
The a reducing sugar is any sugar that is capable of acting as
a reducing agent because it has a free aldehyde group or a
free ketone group. All monosaccharides arereducing sugars, along with
some disaccharides, oligosaccharides, and polysaccharides.
The monosaccharides can be divided into two groups: the aldoses,
which have an aldehyde group, and the ketoses, which have a ketone
group. Ketoses must firsttautomerize to aldoses before they can act as
reducing sugars. The commondietary
monosaccharides galactose, glucose and fructose are all reducing sugars.
Disaccharides are formed from two monosaccharides and can be
classified as either reducing and nonreducing. Nonreducing disaccharides
likesucrose and trehalose have glycosidic bonds between their anomeric
carbons and thus cannot convert to an open-chain form with an aldehyde
group; they are stuck in the cyclic form. Reducing disaccharides
like lactose and maltose have only one of their two anomeric carbons
involved in the glycosidic bond, meaning that they can convert to an openchain form with an aldehyde group.
The aldehyde functional group allows the sugar to act as a reducing
agent, for example in the Tollens' test or Benedict's test. The
cyclic hemiacetal forms of aldoses can open to reveal an aldehyde and
certain ketoses can undergo tautomerization to become aldoses.
However, acetals, including those found in polysaccharide linkages, cannot
easily become free aldehydes.
Reducing sugars react with amino acids in the Maillard reaction, a
series of reaction that occurs while cooking food at high temperatures and
that is important in determining the flavor of food. Also, the levels of reducing
sugars in wine, juice, and sugarcane are indicative of the quality of these
food products.
3,5-Dinitrosalicylicacid (DNS or DNSA, IUPAC name 2-hydroxy-3,5dinitrobenzoic acid)
is an aromatic compound that reacts
with reducing sugars and other reducing
molecules to form 3-amino-5-nitrosalicylic
acid, which absorbs light strongly at 540 nm.
It was first introduced as a method to detect
reducing substances in urine and has since been widely used, for example,
for quantifying carbohydrates levels in blood. It is mainly used in assay
of alpha-amylase. However, enzymatic methods are usually preferred due to
DNS lack of
specificity
Lesson 2 : Nucleic acid
In the 1920's nucleic acids were found to be major components of
chromosomes, small gene-carrying bodies in the nuclei of complex cells.
Elemental analysis of nucleic acids showed the presence of phosphorus, in
addition to the usual C, H, N and O. Unlike proteins, nucleic acids contained
no sulfur. Complete hydrolysis of chromosomal nucleic acids gave inorganic
phosphate, 2-deoxyribose (a previously unknown sugar) and four different
heterocyclic bases (shown in the following diagram). To reflect the unusual
sugar component, chromosomal nucleic acids are called deoxyribonucleic
acids, abbreviated DNA. Analogous nucleic acids in which the sugar
component is ribose are termed ribonucleic acids, abbreviated RNA. The
acidic character of the nucleic acids was attributed to the phosphoric acid
moiety.
The two monocyclic bases shown here are classified as pyrimidines,
and the two bicyclic bases are purines. Each has at least one N-H site at
which an organic substituent may be attached. They are all polyfunctional
bases, and may exist in tautomeric forms. Base-catalyzed hydrolysis of DNA
gave four nucleoside products, which proved to be N-glycosides of 2'deoxyribose combined with the heterocyclic amines. Structures and names
for these nucleosides will be displayed above by clicking on the heterocyclic
base diagram. The base components are colored green, and the sugar is
black. As noted in the 2'-deoxycytidine structure on the left, the numbering of
the sugar carbons makes use of primed numbers to distinguish them from the
heterocyclic base sites. The corresponding N-glycosides of the common
sugar ribose are the building blocks of RNA, and are named adenosine,
cytidine, guanosine and uridine (a thymidine analog missing the methyl
group). From this evidence, nucleic acids may be formulated as alternating
copolymers of phosphoric acid (P) and nucleosides (N), as shown:
~ P – N – P – N'– P – N''– P – N'''– P – N ~
Centrifuge
A centrifuge is a piece of equipment that puts an object in rotation
around a fixed axis (spins it in a circle), applying a potentially strong force
perpendicular to the axis of spin (outward). The centrifuge works using the
sedimentation, where the centripetal acceleration causes denser substances
and particles to move outward in the radial direction. At the same time,
objects that are less dense are displaced and move to the center. In a
laboratory centrifuge that uses sample tubes, the radial acceleration causes
denser particles to settle to the bottom of the tube, while low-density
substances rise to the top.
There are 3 types of centrifuge designed for different applications.
Industrial scale centrifuges are commonly used in manufacturing and waste
processing to sediment suspended solids, or to separate immiscible liquid.
Large centrifuges are used to simulate high gravity or acceleration
environments (for example, high-G training for test pilots). Medium-sized
centrifuges are used in washing machines and at some swimming pools to
wring water out of fabrics.
Figure 1 Centrifuge
Figure 2 tube figure after
centrifugation
Auto pipette
Auto pipette is a mechanical pipette that can transfer measured
amounts of a liquid automatically. It has many capacities and can be
distinguish by the color and the number of capacity written on the body.
Figure 3 auto pipette
The auto pipette must be used with its size tip to pipette the solution.
To protect the solution from contamination, the tip must be changed every
time before pipette the new solution. In this experiment to pipette the solution
from the microcentrifuge tube it should be like the picture below:
Figure 4 How to pipette from micro tube
Gel electrophoresis
Gel electrophoresis is a laboratory method used to separate mixtures
of DNA, RNA, or proteins according to molecular size. In gel electrophoresis,
the molecules to be separated are pushed by an electrical field through a
gel that contains small pores. The molecules travel through the pores in the
gel at a speed that is inversely related to their lengths. This means that a
small DNA molecule will travel a greater distance through the gel than will a
larger DNA molecule.
As previously mentioned, gel electrophoresis involves an electrical
field; in particular, this field is applied such that one end of the gel has a
positive charge and the other end has a negative charge. Because DNA and
RNA are negatively charged molecules, they will be pulled toward the
positively charged end of the gel. Proteins, however, are not negatively
charged; thus, when researchers want to separate proteins using gel
electrophoresis, they must first mix the proteins with a detergent called
sodium dodecyl sulfate. This treatment makes the proteins unfold into a
linear shape and coats them with a negative charge, which allows them to
migrate toward the positive end of the gel and be separated. Finally, after
the DNA, RNA, or protein molecules have been separated using gel
electrophoresis, bands representing molecules of different sizes can be
detected.
Figure 5 gel electrophoresis
Lesson 3 : Enzyme
Enzymes are biological molecules (proteins) that act as catalysts and
help complex reactions occur everywhere in life. Let’s say you ate a piece of
meat. Proteases would go to work and help break down the peptide bonds
between the amino acids.
Enzymes are very specific catalysts and usually work to complete one
task. An enzyme that helps digest proteins will not be useful to break down
carbohydrates. Also, you will not find all enzymes everywhere in the body.
That would be inefficient. There are unique enzymes in neural cells, intestinal
cells, and your saliva.
Four Steps of Enzyme Action:
1. The enzyme and the substrate are in the same area. Some situations have
more than one substrate molecule that the enzyme will change.
2. The enzyme grabs on to the substrate at a special area called the active
site. The combination is called the enzyme/substrate complex. Enzymes are
very, very specific and don't just grab on to any molecule. The active site is a
specially shaped area of the enzyme that fits around the substrate. The
active site is like the grasping claw of the robot on the assembly line. It can
only pick up one or two parts.
3. A process called catalysis happens. Catalysis is when the substrate is
changed. It could be broken down or combined with another molecule to
make something new. It will break or build chemical bonds. When done, you
will have the enzyme/products complex.
4. The enzyme releases the product. When the enzyme lets go, it returns to
its original shape. It is then ready to work on another molecule of substrate.
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that
are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured
and the effects of varying the conditions of the reaction are investigated.
Studying an enzyme's kinetics in this way can reveal the catalytic
mechanism of this enzyme, its role in metabolism, how its activity is
controlled, and how a drug or an agonist might inhibit the enzyme.
Enzymes are usually protein molecules that manipulate other molecules —
the enzymes' substrates. These target molecules bind to an enzyme's active
site and are transformed into products through a series of steps known as
the enzymatic mechanism
E + S <——> ES <——> ES*< ——> EP <——> E + P
These mechanisms can be divided into single-substrate and multiplesubstrate mechanisms. Kinetic studies on enzymes that only bind one
substrate, such as triosephosphate isomerase, aim to measure
the affinity with which the enzyme binds this substrate and the turnover rate.
Some other examples of enzymes are phosphofructokinase and hexokinase,
both of which are important for cellular respiration (glycolysis).
When enzymes bind multiple substrates, such as dihydrofolate
reductase (shown right), enzyme kinetics can also show the sequence in
which these substrates bind and the sequence in which products are
released. An example of enzymes that bind a single substrate and release
multiple products are proteases, which cleave one protein substrate into two
polypeptide products. Others join two substrates together, such as DNA
polymerase linking a nucleotide to DNA. Although these mechanisms are
often a complex series of steps, there is typically one rate-determining
step that determines the overall kinetics. This rate-determining step may be
a chemical reaction or a conformational change of the enzyme or substrates,
such as those involved in the release of product(s) from the enzyme.
Knowledge of the enzyme's structure is helpful in interpreting kinetic data.
For example, the structure can suggest how substrates and products bind
during catalysis; what changes occur during the reaction; and even the role
of particular amino acid residues in the mechanism. Some enzymes change
shape significantly during the mechanism; in such cases, it is helpful to
determine the enzyme structure with and without bound substrate analogues
that do not undergo the enzymatic reaction.
Not all biological catalysts are protein enzymes; RNA-based catalysts such
as ribozymes and ribosomes are essential to many cellular functions, such
as RNA splicing and translation. The main difference between ribozymes
and enzymes is that RNA catalysts are composed of nucleotides, whereas
enzymes are composed of amino acids. Ribozymes also perform a more
limited set of reactions, although their reaction mechanisms and kinetics can
be analysed and classified by the same methods.
Single-substrate reactions
Enzymes with single-substrate mechanisms include isomerases such
as triosephosphateisomerase or bisphosphoglycerate mutase,
intramolecular lyases such as adenylate cyclase and the hammerhead
ribozyme, an RNA lyase. However, some enzymes that only have a single
substrate do not fall into this category of mechanisms. Catalase is an
example of this, as the enzyme reacts with a first molecule of hydrogen
peroxide substrate, becomes oxidised and is then reduced by a second
molecule of substrate. Although a single substrate is involved, the existence
of a modified enzyme intermediate means that the mechanism of catalase is
actually a ping–pong mechanism, a type of mechanism that is discussed in
theMulti-substrate reactions section below.
Michaelis–Menten kinetics
Figure 6Reaction of enzyme
A chemical reaction mechanism with or without enzyme catalysis. The
enzyme (E) binds substrate (S) to produceproduct (P).
Figure 7Saturation Curve
Saturation curve for an enzyme reaction showing the relation between
the substrate concentration and reaction rate.
Michaelis–Menten kinetics:
As enzyme-catalysed reactions are saturable, their rate of catalysis
does not show a linear response to increasing substrate. If the initial rate of
the reaction is measured over a range of substrate concentrations (denoted
as [S]), the reaction rate (v) increases as [S] increases, as shown on the
right. However, as [S] gets higher, the enzyme becomes saturated with
substrate and the rate reaches Vmax, the enzyme's maximum rate.
The Michaelis–Menten kinetic model of a single-substrate reaction is shown
on the right. There is an initial bimolecular reaction between the enzyme E
and substrate S to form the enzyme–substrate complex ES but the rate of
enzymatic reaction increase with the increase of the substrate concentration
up to a certain level but then more increase in substrate concentration does
not cause any increase in reaction rate as there no more E remain available
for reacting with S and the rate of reaction become dependent on ES and the
reaction become unimolecular reaction. Although the enzymatic mechanism
for the unimolecular reaction
can be quite complex, there is
typically one rate-determining enzymatic step that allows this reaction to be
modelled as a single catalytic step with an apparent unimolecular rate
constantkcat. If the reaction path proceeds over one or several
intermediates, kcat will be a function of several elementary rate constants,
whereas in the simplest case of a single elementary reaction (e.g. no
intermediates) it will be identical to the elementary unimolecular rate
constant k2. The apparent unimolecular rate constant kcat is also
called turnover number and denotes the maximum number of enzymatic
reactions catalysed per second.
The Michaelis–Menten equation describes how the (initial) reaction
rate v0 depends on the position of the substrate-binding equilibrium and the
rate constant k2.
(Michaelis–Menten equation)
with the constants
This Michaelis–Menten equation is the basis for most single-substrate
enzyme kinetics. Two crucial assumptions underlie this equation (apart from
the general assumption about the mechanism only involving no intermediate
or product inhibition, and there is no allostericity or cooperativity). The first
assumption is
the so-called quasi-steady-state
assumption (or
pseudo-steady-state hypothesis), namely that
the concentration of the substrate-bound enzyme (and hence also the
unbound enzyme) changes much more slowly than those of the product and
substrate and thus the change over time of the complex can be set to zero .
The second assumption is that the total enzyme concentration does not
change over time, thus
.
The Michaelis constant KM is experimentally defined as the
concentration at which the rate of the enzyme reaction is half Vmax, which can
be verified by substituting [S] = Km into the Michaelis–Menten equation and
can also be seen graphically. If the rate-determining enzymatic step is slow
compared to substrate dissociation (
), the Michaelis constant KMis
roughly the dissociation constant KD of the ES complex.
If is small compared to
then the
term
and also very little ES complex is formed,
thus
. Therefore, the rate of product formation is
Thus the product formation rate depends on the enzyme concentration as
well as on the substrate concentration, the equation resembles a bimolecular
reaction with a corresponding pseudo-second order rate constant
.
This constant is a measure of catalytic efficiency. The most efficient enzymes
reach a
in the range of 108 – 1010 M−1 s−1. These enzymes are so
efficient they effectively catalyse a reaction each time they encounter a
substrate molecule and have thus reached an upper theoretical limit for
efficiency (diffusion limit); and are sometimes referred to as kinetically
perfect enzymes.
Linear plots of the Michaelis–Menten equation
See also: Lineweaver–Burk plot and Eadie-Hofstee diagram
Figure 8Lineweaver
Lineweaver–Burk or double-reciprocal plot of kinetic data, showing the
significance of the axis intercepts and gradient.
The plot of v versus [S] above is not linear; although initially linear at
low [S], it bends over to saturate at high [S]. Before the modern era
of nonlinear curve-fitting on computers, this nonlinearity could make it
difficult to estimate KM and Vmaxaccurately. Therefore, several researchers
developed linearisations of the Michaelis–Menten equation, such as
theLineweaver–Burk plot, the Eadie–Hofstee diagram and the Hanes–Woolf
plot. All of these linear representations can be useful for visualising data, but
none should be used to determine kinetic parameters, as computer software
is readily available that allows for more accurate determination by nonlinear
regression methods.
The Lineweaver–Burk plot or double reciprocal plot is a common way
of illustrating kinetic data. This is produced by taking the reciprocal of both
sides of the Michaelis–Menten equation. As shown on the right, this is a
linear form of the Michaelis–Menten equation and produces a straight line
with the equation y = mx + c with a y-intercept equivalent to 1/Vmax and an xintercept of the graph representing −1/KM.
Naturally, no experimental values can be taken at negative 1/[S]; the
lower limiting value 1/[S] = 0 (the y-intercept) corresponds to an infinite
substrate concentration, where 1/v=1/Vmax as shown at the right; thus,
the x-intercept is an extrapolation of the experimental data taken at
positive concentrations. More generally, the Lineweaver–Burk plot skews
the importance of measurements taken at low substrate concentrations
and, thus, can yield inaccurate estimates ofVmax and KM. A more accurate
linear plotting method is the Eadie-Hofstee plot. In this case, v is plotted
against v/[S]. In the third common linear representation, the Hanes-Woolf
plot, [S]/v is plotted against [S]. In general, data normalisation can help
diminish the amount of experimental work and can increase the reliability
of the output, and is suitable for both graphical and numerical analysis.
Lesson 4 : Glucose Fermentation of yeast
Ethanol fermentation, also called alcoholic fermentation, is a biological
process which converts sugars such as glucose, fructose, and sucrose into
cellular energy, producing ethanol and carbon dioxide as a side-effect.
Because yeasts perform this conversion in the absence of oxygen, alcoholic
fermentation is considered an anaerobic process.
Ethanol fermentation has many uses, including the production of
alcoholic beverages, the production of ethanol fuel, and bread baking.
Alcoholic beverages
Primary fermentation cellar, Budweiser Brewery, Fort
Collins, Colorado
All ethanol contained in alcoholic beverages (including ethanol produced
by carbonic maceration) is produced by means of fermentation induced by
yeast.
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Wine is produced by fermentation of the natural sugars present in
grapes; cider and perry are produced by similar fermentation of
natural sugar in apples and pears, respectively; and other fruit wines
are produced from the fermentation of the sugars in any other kinds of
fruit. Brandy and eaux de vie (e.g. slivovitz) are produced by
distillation of these fruit-fermented beverages.
Mead is produced by fermentation of the natural sugars present in
honey.
Beer, whiskey, and vodka are produced by fermentation of grain
starches that have been converted to sugar by the enzyme amylase,
which is present in grain kernels that have been malted (i.e.
germinated). Other sources of starch (e.g. potatoes and unmalted
grain) may be added to the mixture, as the amylase will act on those
starches as well. Whiskey and vodka are also distilled; gin and related
beverages are produced by the addition of flavoring agents to a
vodka-like feedstock during distillation.
Rice wines (including sake) are produced by the fermentation of grain
starches converted to sugar by the mold Aspergillus oryzae. Baijiu,
soju, and shōchū are distilled from the product of such fermentation.
Rum and some other beverages are produced by fermentation and
distillation of sugarcane. Rum is usually produced from the sugarcane
product molasses.
In all cases, fermentation must take place in a vessel that allows carbon
dioxide to escape but prevents outside air from coming in. This is because
exposure to oxygen would prevent the formation of ethanol, while a buildup
of carbon dioxide creates a risk the vessel will rupture or fail catastrophically,
causing injury and property damage.
Byproducts of fermentation
Ethanol fermentation produces unharvested byproducts such as heat,
carbon dioxide, food for livestock, and water.
In
ethanol fermentation, one glucose molecule breaks down into two pyruvates
(1). The energy from this exothermic reaction is used to bind inorganic
phosphates to ADP and convert NAD+ to NADH. The two pyruvates are then
broken down into two acetaldehydes and give off two CO2 as a waste
product (2). The two acetaldehydes are then converted to two ethanol by
using the H- ions from NADH; converting NADH back into NAD+ (3).