UHOK D

P
O
L
YTECHNIC UNIVERSITY
.TECHNICAL COLLEGE OF ENG
AT PETROCHEMICAL DEPARTMENT

CATALYST LAB REPORT

ENZYME EXPERIMENT
EXP.NO 2
FORTH STAGE

PREPARED BY
MOHAMMED SALAH
REHAN RASHID
BARFI FAQH
FALAK ALI

GROUP :B3
DATE :4/11/2020
:Purpose
.Examine how enzymes are specific to a substrate-1
Observe what happens when the enzyme is denatured by heat -2
.and thus, changing its active site
Investigate the actions of the enzyme and how shape is -3
.important to enzyme reactions
:Abstract
Biotransformation with the help of enzymes can greatly improve
the rate and stereospecificity of reactions in organic chemistry.
However, the use of organic solvents and harsh conditions in
biotechnological applications often correlates with enzyme
deactivation or a dramatic drop-in catalytic activity. Detailed
molecular understanding of the protein structure and
conformational dynamics allows us to address such limitations and
to finely tune catalytic activity by modifying the solvent, the
support, or the active site of the enzyme. Along with physico-
chemical methods of enzyme stabilization, such as additive
approach, chemical modification, and immobilization of enzymes,
approaches of enzyme engineering based on DNA recombination
can be used to enhance the performance of biocatalysts. Since
successful synthetic and industrial applications of biocatalysts
require systems that are not only stable and active, but can also be
reused in a continuous flow process reducing the production cost,
the goal of this chapter is to introduce the reader to the vast scope
of techniques available for enzyme improvement, highlighting their
opportunities and limitations for the real-world technological
processes..
: Introduction
Enzymes are biological catalysts that are believed to be the cornerstones of
life. They assure metabolic needs of cells and assist in a great range of life-
sustaining biochemical reactions. The majority of natural enzymes are
highly efficient and can increase the rate biotransformation up to 1017 fold.
Enzymes can carry their functions at ambit temperatures and pressures,
with a minimum of by-products and waste, leading to the specific product of
interest in a single catalyzed step, whereas synthesis of the same product
with the means of organic chemistry may require many steps and produce a
mixture of undesired isomeric, epimeric, or rearranged compounds . The
field of biotechnology strives to exploit isolated enzymes and whole cell
cultures as biocatalysts capable of accelerating and refining complex
chemical transformations of organic compounds for industrial and synthetic
use. Well known examples of such biocatalysts include microbial lipases that
are used to synthesize cost-effective biopolymers, biodiesel, pharmaceuticals,
and agrochemicals from renewable natural sources, b-glycosidases
employed in industrial plant biomass saccharification and fungal
oxidoreductases that have a potential to become biocatalysts in a bio-based
(circular) economy by converting biomass into renewable building blocks
for manufacturing biodegradable materials . Unfortunately, the scope of
natural enzymes is limited, and certain challenges have to be overcome
before we can rely on biocatalysts for efficient, low-cost industrial
transformations and greener synthetic chemistry. Such challenges include
instability of enzymes in vitro (denaturation in high temperatures or
extreme pH), low selectivity, product and substrate inhibition, and low
reaction yield in non-aqueous solvents. Four general approaches exist to
address the above-mentioned limitations: additive approach, chemical
modification, enzyme immobilization, and protein engineering. While
protein engineering is concerned with modifying functional properties of the
enzyme at the genetic level, the other three approaches are focused on
physico-chemical alterations of the media, enzymatic surface residues, or
support material for biocatalyst stability.
:Result
If these was glucose present mark a positive (+) table. if glucose was
.absent mark a negative (-) the table

+ +

- -

+ +

+ +

- -

:Discussion
:We will answer some question
1-What is an enzyme?
Ans/ Enzymes are biological molecules (typically proteins) that significantly speed up
the rate of virtually all of the chemical reactions that take place within cells. They are
vital for life and serve a wide range of important functions in the body, such as aiding
in digestion and metabolism.
2- What was produced when lactase acted on the sugar lactose?
Ans/ It produces galactose and glucose.

3- How do you know the reaction was successful, turning lactose into glucose
and galactose?

Ans/ the change of glucose test color from teal color to green that mean any
change from teal is indicate positive results.

4- What part of the enzyme got altered when the enzyme lactase was boiled?

Ans/ Lactase is an enzyme that breaks lactose down into glucose and
galactose. In this lab, you will see lactase break lactose down (in skim milk)
into galactose and glucose. You will also observe what happens if the shape
of lactase is changed due to heating. This process is called denaturing.

Also, what happens when the enzyme was boiled? Boiling and Denaturation
At temperatures around boiling, the chemical bonds that hold together the
structure of enzymes begin to break down. The resulting loss of three-
dimensional structure causes enzymes to no longer fit their target substrate
molecules, and enzymes entirely stop functioning.

5- What two conditions can affect the effectiveness of an enzyme?

Ans/ Enzyme activity is dependent on temperature, concentration of enzyme

and concentration of substrates, pH etc. However, the effectiveness of an
enzyme is least affected by the original activation energy of the system. In
other words, enzymes cannot alter feasibility of a reaction but can alter only
rate of a reaction. The enzyme cannot make feasible an infeasible reaction.
Feasibility of a reaction is dependent on original activation energy of the
system.
6-What type of reaction is this? Dehydration synthesis or Hydrolysis?
Explain your answer using these four terms monosaccharide, disaccharide,
water, and bonds.

Ans/ dehydration. the reaction it catalyzes is the hydrolysis of the

disaccharide lactose into the monosaccharide’s galactose and glucose.
Humans require this enzyme for digestion of lactose found in milk and other
dairy products. Lactose, the sugar found in milk, is a disaccharide composed
of glucose and galactose (both six sided sugars). Sucrose, ordinary table
sugar, is also a disaccharide composed of fructose and glucose, Glucose is a
six-sided sugar and fructose is a five-sided sugar.

7- THINK ABOUT IT…If the enzyme lactase breaks down the sugar
lactose, and maltase breaks down maltose; what enzyme is necessary to
break down sucrose?

Ans/ Enzyme sucrase-isomaltose

The SI gene provides instructions for producing the enzyme sucrase-

isomaltose. This enzyme is found in the small intestine and is responsible for
breaking down sucrose and maltose into their simple sugar components.
These simple sugars are then absorbed by the small intestine

8- why can't lactase break down sucrose

Ans/ Lactase is an enzyme that breaks lactose down into galactose and
glucose. Although lactose is similar to sucrose, lactase will break down only
lactose because of the shape of the sugar.
:Conclusions
While enzymes have been involved in commercial production processes for
centuries, their vast potential for a large-scale chemical synthesis and
industrial applications was not fully realized until better empirical models
and methods of biocatalyst stabilization were developed using a trial and
error approach. In this chapter, we reviewed fundamental strategies for
enzyme improvement, such as chemical modification, additive approach,
enzyme immobilization, and protein engineering. It appears that enzyme
immobilization is currently considered to be the most promising strategy for
obtaining industrial biocatalysts with controlled, more specific substrate
interactions, resistance to denaturation, and high product yield at low
cost .At the same time, enzyme engineering methods recently made
numerous successful advances to redesign existing enzymes on the level of
their primary structure using targeted random mutagenesis, in vitro
recombination, and various computational tools. Although there is high
demand for such specialized, robust biocatalysts, they are generally
produced as soluble enzymes, not reusable in the industrial synthesis.
Therefore, integration of physico-chemical methods and protein engineering
is possibly the most efficient strategy for creating a powerful, recyclable
biocatalyst fit for the real-world biotechnological processes.