LITERATURE REVIEW:

The biological degradation of cellulose.

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose,
the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it
forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis.
All organisms known to degrade cellulose efficiently produce a battery of enzymes with
different specificities, which act together in synergism. The study of cellulolytic enzymes at
the molecular level has revealed some of the features that contribute to their activity. In spite
of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases
belong to a restricted number of families. Within each family, available data suggest that the
various enzymes share a common folding pattern, the same catalytic residues, and the same
reaction mechanism, i.e. either single substitution with inversion of configuration or double
substitution resulting in retention of the beta-configuration at the anomeric carbon.
An increasing number of three-dimensional structures is becoming available for
cellulases and xylanases belonging to different families, which will provide paradigms for
molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic
enzymes contain domains not involved in catalysis, but participating in substrate binding,
multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably,
these domains assist in the degradation of crystalline cellulose by preventing the enzymes
from being washed off from the surface of the substrate, by focusing hydrolysis on restricted
areas in which the substrate is synergistically destabilized by multiple cutting events, and by
facilitating recovery of the soluble degradation products by the cellulolytic organism. In most
cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized,
soluble carbon sources and induced in the presence of cellulose.
Induction of cellulases appears to be effected by soluble products generated from
cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are
presumably converted into true inducers by transglycosylation reactions. Several applications
of cellulases or hemicellulases are being developed for textile, food, and paper pulp
processing. These applications are based on the modification of cellulose and hemicellulose
by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented
into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to
reduce emissions of greenhouse gases provides an added incentive for the development of
processes generating fuels from cellulose, a major renewable carbon source.

(Unit de Physiologie Cellulaire, Dpartement des Biotechnologies, Institut Pasteur, Paris, France.)
Sugars are members of the carbohydrate family. Examples include glucose, fructose and
sucrose. Some sugars can act as reducing agents and these sugars will contain an aldehyde
functional group. This property can be used as a basis for the analysis of reducing sugars. For
example Fehlings solution contains copper (II) ions that can be reduced by some sugars to
copper (I) ions. This reaction can be used for the quantitative analysis of reducing sugars. 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 are reducing 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 first tautomerize to aldoses before they can act as
reducing sugars. The common dietary monosaccharides galactose, glucose and fructose are
all reducing sugars.
Disaccharides are formed from two monosaccharides and can be classified as either reducing
or nonreducing. Nonreducing disaccharides like sucrose 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 open-chain 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.
Several qualitative tests are used to detect the presence of reducing sugars. Two of them use
solutions of copper(II) ions: Benedict's reagent (Cu2+ in aqueous sodium citrate) and
Fehling's solution (Cu2+ in aqueous sodium tartrate). The reducing sugar reduces the
copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I)
oxide precipitate. Reducing sugars can also be detected with the addition of Tollen's reagent,
which consist of silver ions (Ag+) in aqueous ammonia.When Tollen's reagent is added to an
aldehyde, it precipitates silver metal, often forming a silver mirror on clean glassware.

3,5-Dinitrosalicylic acid is another test reagent, one that allows quantitative detection. It
reacts with a reducing sugar to form 3-amino-5-nitrosalicylic acid, which can be measured by
spectrophotometry to determine the amount of reducing sugar that was present.

Sugars having acetal or ketal linkages are not reducing sugars, as they do not have free
aldehyde chains. They therefore do not react with any of the reducing-sugar test solutions.
However, a non-reducing sugar can be hydrolyzed using dilute hydrochloric acid. After
hydrolysis and neutralization of the acid, the product may be a reducing sugar that gives
normal reactions with the test solutions.
All carbohydrates respond positively to Molisch's reagent but the test has a faster rate when it
comes to monosaccharides.

CONCEPT OF STANDARD CURVE

A standard curve, also known as a calibration curve, is a type of graph used as a quantitative
research technique. Multiple samples with known properties are measured and graphed,
which then allows the same properties to be determined for unknown samples by
interpolation on the graph. The samples with known properties are the standards, and the
graph is the standard curve.

The spectrophotometer is a routinely used instrument in scientific research.

Spectrophotometry is the quantitative measurement of how much a chemical substance
absorbs light by passing a beam of light through the sample using a spectrophotometer. In this

video, basic concepts in spectrophotometry, including transmittance, absorbance and the

Beer-Lambert Law are reviewed in addition to the components of the spectrophotometer.
These concepts provide a foundation for how to determine the concentration of a solute in
solution that is capable of absorbing light in the ultraviolet and visible range. Furthermore, a
procedure for how to operate the spectrophotometer is demonstrated, including instructions
on how to blank and measure the absorbance of a sample at the desired wavelength. The
video also covers how to make a standard curve for determination of analyte concentration.
Several applications of the spectrophotometer in biological research are discussed, such as
measurement of cell density and determination of chemical reaction rates. Finally, the
microvolume spectrophotometer is introduced, as well as its advantage in measuring the
quality and quantity of protein and nucleic acids.

METHODOLOGY:
1) Standard curve for glucose analysis
i)

Four dilutions of the D-glucose solution (20,40,60,80 mg/ml) prepared. All

dilutions used the acetate buffer provided. These will be your glucose
standard. The dilution calculation was shown in the report.

ii)

1.0 mL from each dilution was taken into a new bottles and then 1.0 mL of
DNS reagent was substituted into it.

iii)

All tubes boiled for exactly 5 minutes in 90 C water-bath and cooled before
adding 3 mL of distilled water.

iv)

OD540 is used to measure the glucose content in all tubes in the UV/Vis
spectrophotometer.

2) Cellulose degradation
i.

three different cellulose degradation trials were given, which were conducted the

ii.

day before the experiment.

2-mL of culture solution was put into a 2-mL centrifuge tube and centrifuge for 5

iii.

min at the highest speed to separate the cells from the solution.
the solution part was pipetted and the reducing sugar content was analyzed.

REFERENCE:
i.
ii.
iii.
iv.

https://en.wikipedia.org/wiki/Reducing_sugar
https://en.wikipedia.org/wiki/Standard_curve
http://www.jove.com/science-education/5038/introduction-to-the-spectrophotometer
1Unit de Physiologie Cellulaire, Dpartement des Biotechnologies, Institut Pasteur,
Paris, France.