Introduction of varying temperatures, pH, and enzyme substrate concentrations.

Introduction

Enzymes are proteins that acts as
catalysts, increasing the rate of chemical reactions. With containing many
different enzymes, enzymes prove to be viable molecules, as they can ultimately
determine the many possible chemical reactions that take place within the cell.1With
an enzyme able bind to the substrate through specificity, the substrate
correctly orients itself binding to the active site. Once positioned, the
enzyme can lower the activation energy of the reaction causing the chemical
reaction to occur at a rapid pace. The efficiency of an enzyme can vary
depending on the environment. Some factors may include temperature, pH, and
differences in enzyme substrate concentration.

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Cellobiase is an enzyme involved in
the final steps of breaking down cellulose to glucose, as the cellobiase enzyme
hydrolyzes the beta-glucosidic linkage of a cellobiose molecule to obtain two
beta-D-glucose molecules.2 This process is used by fungi and
bacteria to produce glucose as a food source with cellobiase catalyzing
p-nitrophenyl glucopyranoside into glucose and p-nitrophenol. This process of
breaking down cellulose can also be seen in converting cellulose to ethanol and
other chemical products in a multistep process.3 The steps include:
pretreatment, hydrolyzing the polymers, converting many of the smaller
metabolic molecules, and separating and purifying the desired products.3

The purpose of this lab is to
determine the rate of enzymatic activity of cellobiase over time under the
effects of varying temperatures, pH, and enzyme substrate concentrations.

Composed of five different experiments, each experiment highlights each
external factor listed. In this study, I believe cellobiase will thrive in
moderate temperatures and pH levels, whereas low and high temperatures and pH
levels will denature or prevent cellobiase from working to the best of its
ability.

 

Cellobiase is an enzyme which hydrolyzes the
8-glucosidic linkage

of  a 
cellobiose
molecule  to give  two molecules of  P-D-glucose

Cellobiase is an enzyme which hydrolyzes the
8-glucosidic linkage

of  a 
cellobiose
molecule  to give  two molecules of  P-D-glucose

Materials and
Methods

Determining the standard curve

Five conical tubes were labeled
S1-S5. Deionized water was added to tubes S2 through S5. 1 mM standard (p-nitrophenol)
and deionized water was added, and serial dilution was performed. 1X stop
solution (0.1 M Carbonate buffer, pH 9.5) was added to each tube. The
absorbance of each standard was measured at 410 nm

Determining the reaction rate in
the presence or absence of enzyme

1X Stop solution (0.1 M Carbonate
buffer, pH 9.5) was added into each microcentrifuge tube. A reaction tube and
control tube were prepared by adding differing amounts of 1.5 mM substrate
(p-nitrophenyl glucopyranoside) into different conical tubes. 10X Resuspension
buffer (Sodium Acetate, pH 5) was added into the control conical tube. This
solution containing 10X Resuspension buffer (Sodium Acetate, pH 5) and 1.5 mM
substrate (p-nitrophenyl glucopyranoside) is added to the start microcentrifuge
tube. Low concentration enzyme solution (cellobiase) was added to the conical
reaction tube. The solution from the reaction tube was removed and added into
five microcentrifuge tubes E1 through E5 with 1X stop solution (0.1M Carbonate
buffer, pH 9.5) at 1 m, 2 m, 4 m, 6 m, and 8 m respectively. The solution from
the control tube was added into the stop microcentrifuge tube. The absorbance
of each standard was measured at 410 nm.

Determining the effect of
temperature on the reaction rate

Low concentration enzyme solution
(cellobiase) was added to each appropriately labeled microcentrifuge tubes and 1.5
mM Substrate (p-nitrophenyl glucopyranoside) was added to each appropriately labeled
microcentrifuge tubes. The 0 °C Enzyme and Substrate tubes were placed in an
ice bucket for 5 m. The 22 °C Enzyme and Substrate tubes were incubated at RT
for 5 m. The 37 °C Enzyme and Substrate tubes were placed in a heat block for 5
m. 1X Stop solution (0.1 M Carbonate buffer, pH 9.5) was added into three new
microcentrifuge tubes. Each of the Low Concentration Enzyme Solution
(cellobiase) tubes were added into the appropriately labeled Substrate tubes
consisting the 1.5 mM Substrate (p-nitrophenyl glucopyranoside) at the
appropriate temperatures. The reactions in each of the Enzyme and Substrate
tubes were added in to each of the 1X Stop solution (0.1 M Carbonate buffer, pH
9.5) after 2 m. The tubes were incubated at RT for 5 m. The absorbance of each
solution was measured at 410 nm.

Determining the effect of pH on the
reaction rate

1X Stop solution (0.1 M Carbonate
buffer, pH 9.5) was added to each microcentrifuge tube. 1X Resuspension buffer
(3.0 M Sodium Acetate, pH 5) was added into the pH 5 Buffer microcentrifuge tube,
1X Stop solution (0.1 M Carbonate buffer, pH 9.5) was added into the pH 8.6
Buffer microcentrifuge tube, and a mixture of 1X Stop solution (0.1 M Carbonate
buffer, pH 9.5) and deionized water was added into the pH 6.3 Buffer microcentrifuge
tube. 3.0 mM Substrate (p-nitrophenyl glucopyranoside) was added into each of
the microcentrifuge Buffer tubes. The Low concentration enzyme solution
(cellobiase) was added into each microcentrifuge Buffer tubes. Each of the
reactions in the microcentrifuge Buffer tubes were added to the appropriately
labeled 1X Stop solution microcentrifuge tubes. The absorbance of each solution
was measured at 410 nm.

Preparing the Mushroom Extract

1 g of mushroom (Bio-Rad, as per
manufacturer’s instructions) was homogenized with 2 mL of 1X Extraction buffer
(3.0 M Tris, Magnesium Chloride, Triton X-100, pH 7.2) for every gram. The
mushroom was grinded to produce a slurry. The slurry was added in a
microcentrifuge tube and spun at 14,000 rpm for 2 m. 

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