CHM161L – E01

4Q 2018 – 2019
Instructor: Dr. Dahlia C. Apodaca

Proteases in Protein Catabolism

Matienzo, Miguel F.
BECM, Mapua University
Cainta, Rizal
miguelmatienzo9798@gmail.com

Abstract

Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide
bonds in proteins. They can be found in all living organisms, from viruses to animals and humans.
Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological
processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several
sectors of industry and biotechnology, furthermore, numerous research applications require their use,
including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during
nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody
fragments for research, diagnostics and therapy, exploration of the structure-function relationships by
structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide
sequencing and proteolytic digestion of proteins in proteomics. The objectives were half met wherein Apple
and Chinese cabbage are the source of sample. The researchers were able to observe the effect of protease
activity while the effects of temperature on protease activity were not due to unavailability of reagent.

Keywords: Proteolytic enzyme, hydrolyze, synthesis, recombinant antibody, and protease

1. Introduction

Digestion is the process which helps an organism to convert food molecules into forms that are nutritionally
useful to that organism. In the case of animals possessing an alimentary canal, numerous extracellular
enzymes are secreted into the lumen of this structure so that ingested food items may be transformed into
forms that can be absorbed through the epithelium. It has been established that fishes and higher vertebrates
utilize the same enzymes and hormones in the breakdown of proteins into amino acids. In fish, the specific
levels of digestive enzyme activities are dependent on age, diet, season, and/or ambient temperature.
Proteases attack peptide bonds of proteins and polypeptides, thereby converting large peptide chains into
shorter polypeptide segments. There are two basic types of proteases, the endopeptidases and the
exopeptidases. In case of former, the enzyme attacks bonds located deep within the substrate protein, thus
transforming large peptide chains into shorter peptide segments. Exopeptidases function by converting

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Instructor: Dr. Dahlia C. Apodaca

these shorter segments, located near the end of a peptide chain, into free amino acids, dipeptides and
tripeptides. Trypsin, chymotrypsin and elastase are endopeptidases occurring in the alimentary canal of
perhaps virtually all invertebrates and vertebrates possessing this digestive structure. The degradation of
proteins is important for plant development. Proteins may become unwanted because they have become
damaged, or the requirement of the cell has changed making previously synthesized proteins redundant. In
several key stages of plant development such as during germination of seeds and during leaf senescence,
storage and other proteins are degraded to release amino acids for transport to the most actively growing
regions of the plants. Protease activity is low in the youngest leaves and very high in old senescing
leaves.(Koak, Kim, Choi, Baik, & Kim, 2011)

Protease refers to a group of enzymes whose catalytic function is to hydrolyze peptide bonds of proteins.
They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various
peptide bonds. Proteases also have many functions. The action of proteases was believed to be restricted to
digestive purposes, extracellular modeling and/or remodeling of tissues, mainly through proteolytic activity
on interstitial molecules, occurring throughout homeostasis and development or, in aberrant maladaptive
circumstances, during disease pathogenesis.(Yamauchi & Kaminogawa, 1972)

Figure 1. Hydrolysis of peptide bond

The researchers deem to conduct an experiment wherein the concept behind would be focused on. The
objectives are to observe the effect of protease activity and to determine the effects of temperature on
protease activity.

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4Q 2018 – 2019
Instructor: Dr. Dahlia C. Apodaca

2. Methodology

2.1 Chemical Reagents, Samples, and Apparatuses

The reagents and apparatuses used for experiment 3 are protease solution, gelatin powder, phosphate buffer,
milk powder, distilled water, nihydrin solution, hot plate, centrifuge machine, and glassware.

2.2 Protease activity in fruits and vegetables

The fruit and vegetable samples were cut into same sizes and was placed inside a 20mL test tube. It was
then labeled according to sample. A gelatin solution was prepared and was placed inside the test tube. It
was then incubated in an ice bath for 5 minutes. Observations were then made. Phosphate buffer was added
after and was heated in a water bat for 80 degree Celsius. 5 drops of ninhydrin solution was added and was
placed in water bath for another 10 minutes. The presence of precipitate was then noted.

2.3 Effects of Temperature on Protease Activity

5 test tubes containing 2 mL of protease solution were prepared. An X mark was drawn beneath the 50 mL
beakers and was then marked according to their respective temperature. A milk solution was prepared by
dissolving 10 grams of milk powder and 100mL distilled water. 10mL of the milk solution were placed
inside the beaker together with the test tube and was then heated according to the labeled temperature of
the beaker. After 5 minutes, the enzyme and the milk were then mixed together. Observations were then
made after.

3. Results and Discussion

Protease Activity in fruits and vegetables. The sample used by the researcher in protease activity analysis
are fruit: Apple, and vegetable: Chinese cabbage. As observed in the data obtained in table 1, both sample
were deemed to contain low proteolytic activity with no reaction occurring upon the addition of the
Nihydrin solution. To prove the data obtained, the researcher looked for an alternate data that would accept
the observation made.

Table 1. Protease activity in chosen fruit and vegetable

Sample Viscosity Volume of precipitation (very small amount) Color

Apple 1 1 No color
Chinese Cabbage 1 2 No color

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Table 2. Protease activity of various sample experimented

Fruit Color Vegetable Color

Banana Strong Blue Sweet potato Light Blue
Mango Light Blue Cabbage No color
Guava Light Blue Jute leaves (Saluyot) No color
Pineapple Strong Blue Talbos Light Blue
Kiwi Light Blue Malunggay No color

In the results obtained by the colleagues of the researcher, banana, mango, guava, kiwi, pineapple and
potato portrayed observations the propose the presence of protease activity. This is deem to be true such
that corresponding protease in these fruits are for industrial use. (Kiwifruit, mango and banana), and
pineapple contains the protease, actinidin and bromelain, respectively. It functions as a dietary supplement
and wound treatment. Guava contains papain which is used for digestive aid especially for treating parasitic
worms.

Figure 2. Proteolytic Activity of various fruit and vegetable samples (Yamaguchi, Yamashita, Takeda, &
Kiso, 1982)

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Instructor: Dr. Dahlia C. Apodaca

As shown in figure 1, apple and Chinese cabbage are observed to contain no or less proteolytic activity.
Similar procedure was made by making use of the gelatinization method. Because of such, the data observed
by the researcher proved to be true.

The ninhydrin test was used because it is a powerful oxidizing agent and its presence, amino acid undergo
oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin
(hydrindantin). The NH3 formed from a amino group reacts with another molecule of ninhydrin and is
reduced product ( hydrindatin) to give a blue substance diketohydrin (Ruhemanns complex). Thus used for
the test in protein.

Scheme 1. Nihydrin test reaction

Effects of Temperature on Protease Activity. The researchers were not able to obtain the data due to lack
of reagent. According to principle, Higher temperatures speed up the effect of enzyme activity, while lower
temperatures decrease the rate of an enzyme reaction. At higher temperatures, more molecules collide,
increasing the chance that an enzyme will collide with its substrate. However, if the temperature is too high,
an enzyme will denature, which causes the shape of the enzyme to change. If the enzyme’s shape changes,
it cannot bind to the substrate. The role of the protease enzyme is to break the protein in milk allowing the
x mark to be visible.

Temperature Observation
20 °C
30 °C Due to lack of protease enzyme, no reaction
40 °C occurred. Thus, the X mark was observed to be
50 °C not visible.
60 °C

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4Q 2018 – 2019
Instructor: Dr. Dahlia C. Apodaca

4.Conclusion and Recommendation

The objective was partly achieved as the researchers were able to observe the effect of protease activity of
the respective fruit and vegetable samples on gelation. For the effect of temperature on protease activity,
the researchers were not able to conduct the experiment due to lack of reagent. It was observed that fruit
samples contain more protease enzyme than the vegetable sample. Error may have been made upon
conducting the experiment and to provide consistency with the data, the researchers recommend to carefully
protocol to obtain accurate results.

3. References

Koak, J. H., Kim, H. S., Choi, Y. J., Baik, M. Y., & Kim, B. Y. (2011). Characterization of a protease
from over-matured fruits and development of a tenderizer using an optimization technique. Food
Science and Biotechnology, 20(2), 485–490. https://doi.org/10.1007/s10068-011-0067-9
Yamaguchi, T., Yamashita, Y., Takeda, I., & Kiso, H. (1982). Proteolytic enzymes in green asparagus,
kiwi fruit and miut: Occurrence and partial characterization. Agricultural and Biological Chemistry,
46(8), 1983–1986. https://doi.org/10.1080/00021369.1982.10865376
Yamauchi, K., & Kaminogawa, S. (1972). Decomposition of milk proteins by milk protease. Agricultural
and Biological Chemistry, 36(2), 249–254. https://doi.org/10.1080/00021369.1972.10860237

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