NUS High School of Math and Science

Class M10602 and M10606

Module BL 6402 Proteins
Free Research Project Proposal

Title: Comparison of the amyloid-degrading activities of nattokinase from Bacillus subtilis natto
extracted from three different sources

Students: Beryl Yee Wen-Yen, Chew Xizhi Stephanie, Dorothy Hannah Huang Min, Lin Fang Yi,
Royston Ong

Summary:

Introduction and Literature Review:

Natto is a traditional Japanese food made from fermented soybeans. The dish has a pungent,
cheese-like smell and a strong unami taste, and is known for its thick, sticky texture. In the glue-
like substance surrounding the soybeans in natto, the enzyme nattokinase produced by Bacilus
subtilis natto was found, in 1980, by Dr “Natto” Hideyuki Sumi, have anti-blood-clotting
capabilities.

Ensuing research characterized nattokinase as a subtilisin-like serine protease, an enzyme that

breaks down proteins with the amino-acid serine in its active sites. Nattokinase is a polypeptide
275 amino acids in length and has a molecular weight of 27,728 g/mol. The enzyme was found
remarkable because of its fibrinolytic properties.

After injury or trauma, in particular to epithelial cells of blood vessels, the protein fibrin forms what
is commonly known as a “blood clot”. Foreign bodies in the blood such as bacteria and fungi can
further stimulate the formation of cross-linked fibrin in an inflammatory response. The blood clots
formed, when there cease to be a threat of blood loss, will travel to other parts of the body via the
blood stream if not broken down by plasmin. This blood clot, known as thrombus, is the major
cause of diseases such as cerebral hemorrhage, cerebral infarction, cardiac infarction, angina
pectoris, as well as certain types of dementia and diabetes. Several studies both in vitro and in
vivo have confirmed the fibrinolytic capabilities of nattokinase, even though there has been
insufficient research in clinical usage of nattokinase, and it has instead been commercialized as
an alternative medicine.

Recent research has shown that nattokinase from Bacillus subtilis natto is also capable of
amyloid degradation. Amyloid is an insoluble fibrous protein which when accumulated in the body
will lead to diseases such as Alzheimer’s Disease, Parkinson’s Disease and Diabetes mellitus
type 2 and several prion diseases. Nattokinase extracted from commercial products was found to
demonstrate significant amyloid-degrading abilities, more than that of trypsin and plasmin, though
less than proteinase K and subtilisin Carlsberg at 37ºC and at pH 7.4 (in order to simulate human
body conditions). An alkaline protease, nattokinase was found to have optimal activity at pH 10 at
a temperature of 40ºC, and remains functional from neutral to alkaline pH, but loses functionality
at pH 5 and below. At a constant pH of 7, the amyloid-degrading activities of nattokinase are
optimal at the temperature of 50ºC, and loses its amyloid-degrading abilities above 60ºC. Since
nattokinase is known to be functional in fibrinolysis when administered orally, it could provide
therapy methods for amyloid-related diseases. Furthermore, since natto is a healthy, popular
food, promotion of the food might decrease the incidences of amyloid-diseases as well. However,
more research needs to be done on the amyloid-degrading abilities of nattokinase to date.

Studies have shown that nattokinase can be produced by fermentation on various substrates
instead of direct extraction from readily available commercially produced Natto. The nattokinase
from these substrates produce similar fibrinolytic abilities as the commercial Natto-extracted
nattokinase. Available on the market are also nattokinase tablets in which the protease is already
in its purified form, ready for use. However, little research has been done on whether the
nattokinase from other substrates will produce similar amyloid-degrading abilities to the
nattokinase extracted from commercial Natto or nattokinase tablets. Soybean curd residue (SCR)
and Wet corn distiller’s grain (WGD) are two substrates that had been used to ferment and
produce nattokinase in a previous study. These substrates are waste products from the food
manufacturing industry and recycling the substrates can provide an eco-friendly source of
nattokinase for further studies in the amyloid-degrading capabilities of nattokinase if the
nattokinase from these substrates are similar to commercially extracted or tablet nattokinase.
This could encourage more research in the area and also promote green experiments.

Purpose:

The purpose of this study is to determine if the amyloid-degrading abilities of nattokinase

fermented from Soybean curd residue and Wet corn distiller’s grain are similar to that of
nattokinase extracted from commercial Natto and Natto tablets.

Rationale: I have no idea what the rationale is, seriously.

Hypotheses: That all the substrate have the same amyloid-degrading ability LAR. But state the
null hypotheses (that there is no significant difference between any of them in amyloid-degrading
ability) in a nice paragraph=)

Delimitations: The results of this study can only show that the amyloid-degrading ability of the
nattokinase from recycled substrate is similar to that of nattokinase extracted from commercial
Natto and Natto tablets. It does not indicate anything about the amyloid-degrading ability in vivo
and the results of the study can only form the basis of further research but not support further
studies on the amyloid-degradation effects of nattokinase in humans or other animals.

Definitions: I dunno what there is to define but I guess can use the handout on the assignments
as a guide??

Methods: Under the folder “the Rest of FRP”, the file named amyloid-degrading is the main
paper that can be used for methods of measuring amyloid-degrading activities. The file named
“Thrombolytic Activities of….” Can be used for finding methods of extracting nattokinase from the
commercial Natto, and natto fermented on the respective substrates. Please also include
“optimization of nattokinase from various substrates” in the procedures.

For analysis just say we will use ANOVA or something. HAHAHA

Subjects:

Materials:

Procedures:

References:

Proposed Budget: Under the folder Money Matters there is the price of nattokinase tablets as
well as local Japanese market natto prices