Indonesian Journal of Medical Laboratory Science and Technology
Open Access

p-ISSN 2684-6748
e-ISSN 2656-9825

RESEARCH ARTICLE

Exploration and molecular identification of proteolytic bacteria
from rusip pacific oyster (Crassostrea gigas) as anticoagulant
agent candidates
Muhammad Ardi Afriansyah
Gusti Dimas Refian Akbar

1, Sudarwin

1, Sri Sinto Dewi

1,

1

1Medical Laboratory Technology Study Program, Universitas Muhammadiyah Semarang, Semarang, Indonesia

Correspondence:
M. Ardi Afriansyah
Medical Laboratory Technology Study
Program, Universitas Muhammadiyah
Semarang, Semarang – 50273, Indonesia

Email: afriansyah@unimus.ac.id
Article history:
Received: 2024-06-20
Revised: 2024-12-21
Accepted: 2025-01-14
Available online: 2025-04-30

Keywords:
Anticoagulant
Protease
Rusip fermentation
Proteolytic bacteria
Pacific oysters
https://doi.org/10.33086/ijmlst.v7i1.6083

1.

Abstract

The marine symbiont Staphylococcus epidermidis strain CGF-6, a proteaseproducing bacterium, has been successfully isolated from Rusip Pacific Oyster
(Crassostrea gigas). S. epidermidis is a non-spore-forming, Gram-positive
coccus commonly found in marine environments due to their ability to tolerate
high salinity. The aim of this study was to identify proteolytic bacteria from
Rusip fermented C. gigas as potential candidates for the development of
anticoagulant agents. Bacterial isolation was performed through the
fermentation process of Rusip. After seven days, bacterial colonies were
purified three times using Nutrient Agar. The selection of proteolytic bacterial
was conducted qualitatively using a skim milk agar medium. The bacterial
isolates exhibiting the highest protease activity were identified through 16S
rRNA gene sequencing using universal
` primers Bact 27F and UniB 1492R.
Phylogenetic tree analysis, conducted with the MEGA X program, helped
determine the relationships between species. Out of the 18 bacterial isolates
obtained from the Rusip fermentation of C. gigas, three isolates (CGF-1, CGF-6,
and CGF-11) exhibited hydrolysis zones around their colonies on skim milk
agar, indicating protease activity. Among these, isolate CGF-6 showed the
highest proteolytic index of 0.5 and was identified as Staphylococcus
epidermidis strain CGF-6. S. epidermidis strain CGF-6 has the potential to serve
as a valuable source of protease production for the development of
anticoagulant agents. However, further studies, including enzyme
characterisation, optimisation, and both in vitro and in vivo anticoagulant
activity tests, are necessary to assess the efficacy and safety of this enzyme as
a candidate for anticoagulant agents.

INTRODUCTION

Protease enzymes can be derived from various sources, including microorganisms, plants, and animals found in
both marine and terrestrial ecosystems. Recent studies have shown promising results in exploring protease-producing
marine microbes. Specifically, researchers have successfully isolated protease-producing bacteria from marine
organisms such as sand sea cucumbers (1), brown algae (2), seaweed (3), and nudibranch (4). One noteworthy example is
the Pacific oysters (Crassostrea gigas), a bivalve with a soft body protected by two shell valves. These oysters have a
relatively high protein content. ranging from 50% to 56%. Due to their ecological and economic significance, oysters
provide numerous benefits (5). They have extensive industrial applications, including functional foods, natural
pharmaceuticals, and valuable bioactive compounds. The bioactive components found in Pacific oysters exhibit various
activities, such as anticoagulant, antioxidant, anti-inflammatory, antimicrobial, anticancer, antihypertensive, and
immunomodulatory effects (6). The abundance of these bioactive compounds, particularly proteins, along with other
essential nutrients like minerals, glycogen, essential amino acids, and fatty acids, makes Pacific oysters a promising natural
resource for cultivating proteolytic bacteria (7).
Citation: Afriansyah MA, Sundarwin S, Dewi SS, Akbar GDR. Exploration and molecular identification of proteolytic bacteria from rusip
pacific oyster (Crassostrea gigas) as anticoagulant agent candidates. Indones J Med Lab Sci Technol. 2025;7(1):70–79.
https://doi.org/10.33086/ijmlst.v7ii.6083
This is an open access article distributed under the Creative Commons Attribution-ShareAlike 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. ©2025 The Author(s).
https://journal2.unusa.ac.id/index.php/IJMLST

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Proteases obtained from marine environments generally exhibit greater activity compared to those sourced from
terrestrial environments. The complexity of the marine environment, characterised by high salinity, high pressure, low
temperature, and unique light conditions, contributes to the distinct characteristics of enzymes produced by marine and
terrestrial microorganisms (8). Utilizing marine symbiotic microorganisms for protease enzyme production is
advantageous due to the suitability of these complex environments for harsh industrial processes. This has led to the recent
development of marine microbial enzyme technology, resulting in enzyme products that are used in pharmaceuticals, food
additives, and fine chemicals (9). Furthermore, using microorganisms as sources of protease offers notable flexibility. Their
ability to be propagated through colony breeding allows for large-scale and continuous production with relatively short
production cycles. Additionally, the potential for genetic manipulation enables the optimisation of protease enzyme
production (10).
Proteolytic bacteria typically belong to genera such as Staphylococcus, Streptococcus, Bacillus, Pseudomonas, and
Proteus. These species are commonly found in marine environments, as they thrive in high-salinity conditions and possess
inherent proteolytic capabilities. They produce protease enzymes extracellularly as secondary metabolites and are
generally non-pathogenic (11). Proteases have widespread applications across various industries, especially those that
utilize enzyme-based technologies. However, while employing broad-spectrum enzymes like proteases holds great
potential, it comes with drawbacks, such as susceptibility to degradation and high production costs (12). Moreover, the
clinical application of enzymes must adhere to regulatory requirements, including being free from toxic substances and
maintaining high purity level. Numerous protease enzymes that serve as anticoagulants have received approval from the
Food and Drug Administration (FDA). Nonetheless, there is an ongoing need for novel therapeutic enzymes to address
emerging medical challenges (13).
The search for anticoagulant agents derived from natural sources remains an active area of research, driven by the
need for safe and effective anticoagulants with minimal adverse effects on the human body. Currently available
anticoagulants often require high dosages and may lead to potential complications. The growing acceptance of using
protease-producing bacteria in industrial settings to develop eco-friendly enzymes has spurred increased interest in
exploring bacterial strains capable of producing proteases with anticoagulant potential (14). Ongoing research indicates
that proteolytic bacteria should be isolated from marine biota rich in bioactive compounds that act as anticoagulants. When
selecting candidate sources of protease producers for anticoagulants, several criteria must be considered: the capacity to
produce protease, stability, viability, and non-toxicity. Additionally, both in vitro and in vivo studies are crucial for assessing
the anticoagulant activity of the produced proteases. These studies evaluate enzyme pathogenicity and determine the
efficacy of potential anticoagulant agents (15). The use of marine organisms, such as the Pacific oyster, as a natural resource
rich in microbial diversity has not been fully explored for this purpose. However, the potential for discovering proteaseproducing bacteria is promising, given that most marine microorganisms are non-pathogenic. Therefore, this research aims
to isolate and identify proteolytic bacteria from rusip Pacific oysters as a potential source of protease production for
developing anticoagulant agents.

2.

MATERIALS AND METHODS

2.1. Rusip Fermentation and Bacterial Isolation
Sample of C. gigas were collected from Ngebum Beach, Kaliwungu, Kendal Regency, Central Java, Indonesia.
The morphology and characteristics of the sample were checked based on the description of the species Crassostrea
gigas refers to https://www.fisheries.noaa.gov/species/pacific-oyster. Fresh oyster samples were sterilized using alcohol
70% and sterile distilled water, then crushed using sterile mortar. Rusip fermentation using a mixture of 25% coarse salt
(1.25 grams), and 10% palm sugar (0.5 grams), combined with 5 grams of crushed oyster meat. The mixture was put
into a sterile closed container (anaerobic) and left for 7 days at a temperature of 25ºC. A 1000μL aliquot of the
fermentation product was mixed with sterile physiological NaCl (Cat. No. 3526099002, No Brand, Indonesia) at
concentration ranging from 10-1 – 10-5 and homogenized. Then, 100μL of each dilution was spread onto Zobell Marine Agar
(ZMA) (Cat. No. 2216, HiMedia, India) using a sterile spreader and incubated at 37ºC for 24 hours. ZMA is a specific media
for bacteria from marine environment. Colonies which appear with diverse morphological characteristics in the medium
were then separated on Nutrient Agar (NA) plates (Cat No. M561, HiMedia, India) through three rounds of streaking to
obtain pure colonies (2).

2.2. Morphological Identification
Microscopic identification was carried out using the Gram staining method (Cat. No. 77730-1KT-F, MERCK, USA).
Bacterial colonies growing on the medium were stained using crystal violet, iodine, alcohol, and safraninD. Morphological
identification was observed under a microscope with an 1000x total magnification using an oil immersion lens, focusing on
shape, colour, arrangement and Gram characteristics. Macroscopic identification was based on the colony characteristics
observed on the medium including shape, color, edge, elevation, and consistency (2).

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2.3. Selection of Proteolytic Bacteria
To qualitatively assess protease activity, the pure isolate was cultured on 5% (w/v) Skim Milk Agar (SMA) (Cat. No.
M763, HiMedia, India) and incubated at 37°C for 24-48 hours. The presence of a clear zone surrounding the colony indicated
proteolytic activity. This hydrolysis zone suggests the bacteria possess proteolytic activity, which can be expressed as a
proteolytic index. The determination of proteolytic index can be calculated using the following formula:
Hidrolysis capacity =

clear zone diameter−bacterial colony diameter
clear zone diameter

........................................................................................................................ (1)

The hydrolysis capacity index serves as a reference for assessing the presence of protease activity in millimeters
and the bacteria’s ability to produce protease (2).

2.4. Molecular Identification
2.4.1. 16S rRNA Identification
Bacterial DNA was extracted using the Quick-DNAMagbead Plus Kit (Zymo Research, D4082, US). The purity of the
genomic DNA was assessed with a Nanodrop spectrophotometer (Thermo ScientificTM). A total of 50 µL DNA sample was
measured using Nanodrop spectrophotometer at 260 and 280 nm wavelengths to calculate the absorbance ratio of
A260/280. The Identification of the 16S rRNA gene was conducted at PT. Genetika Science Indonesia. DNA amplification
was performed using Thermal Cycler™ and universal primers Bact 27F (5' AGAGTTGATCCTGGCTCAG 3') and UniB 1492R
(5' GGTTACCTTGTTACGACTT 3') (1). The amplification process employed MyTaq HS Red Mix, 2X (Bioline, BIO-25048, US).
The PCR reaction consisted of 12.5 µL of MyTaq HS Red Mix (2X), 25 µL of double-distilled water (dd H2O), and 50 µL of
CGF-6 DNA with a concentration of 1.96 ng/µL. The PCR protocol was performed for 36 cycles, included denaturation at
95°C for 4 minutes, followed by annealing at 55°C for 35 seconds, and extension at 72°C for 45 seconds. PCR products
(1400bp) were analysed using 1% TBE agarose gel electrophoresis, followed by visualization of amplicons under UV light
at 312 nm.

2.4.2. Phylogenetic Tree Construction
The 16S rRNA gene was sequenced using the Sanger sequencing method. The DNA sequencing results were analysed
using Base Assemble software, processed manually and compared with data from https://blast.ncbi.nlm.nih.gov/Blast.cgi
through the BLAST program (Basic Local Alignment Search-Tool for Nucleotide) to compares sequences against GenBank
database to find the closest matches based in similarity scores. Phylogenetic Tree analysis and design were conducted using
the MEGA X program by Neighbor-joining method to determine the evolutionary relationship between the bacteria.

3.

RESULTS AND DISCUSSION

3.1. Bacterial Exploration from Rusip Pacific Oyster
Pacific oysters (Crassostrea gigas) (Figure 1) have characteristics consistent with the literature (16), including an
elongated "cupped" shaped shell with rough and slightly sharp edges, and a white interior. The bacterial isolation results
from rusip (fermented Pacific oyster products) yielded 18 distinct bacterial isolates, which were successfully purified using
Nutrient Agar medium (Figure 2).
A

B

body tissue
gill
mantle

Figure 1. Pacific oysters (Crassostrea gigas). (A) Shells. (B) Inside the body.

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Approximately 40% of the Pacific oyster's body consist of meat that is protected by the shell (Figure 1A). The shell
is composed of about 95% calcium carbonate (CaCO3) and 5% shell protein. As a result, oyster shells are widely utilized in
health products and environmentally friendly waste processing. In this study, the utilized body part of Crassostrea gigas
was the whole tissue, including body tissue, gills, and mantle (Figure 1B). The body tissue of Pacific oyster contains bioactive
compounds with various biological activities, such as antioxidants, anti-inflammatory, antimicrobial, antihypertensive,
anticancer, and anticoagulants properties. The use of the entire Pacific oyster tissue is further supported by its high protein
content, which can enhance the acquisition of proteolytic microorganisms through the fermentation process (6).

3.2. Proteolytic Bacterial Selection
Three bacterial isolates designated CGF-1, CGF-6, and CGF-11, were isolated from rusip Pacific oyster (Crassostrea
gigas) and exhibited protease hydrolysis activity (Figure 3). Among these isolates, CGF-6 demonstrated the most significant
protease hydrolysis activity, outperforming both CGF-1 and CGF-11. Proteolytic activity was qualitatively assessed on skim
milk agar medium by measuring the size of the proteolytic zone created by the bacterial isolates on the screening medium.
To produce secondary metabolites, including protease enzymes, bacteria require cultivation in a medium containing a
protein substrate like skim milk, which contains casein that readily reacts with these enzymes (17).
A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

K

L

M

Figure 2. 18 pure bacterial isolates associated with rusip Pacific oysters (C. gigas). (A) CGF-1. (B) CGF-2.
(C) CGF-3. (D) CGF-4. (E) CGF-5. (F) CGF-6. (G) CGF-7. (H) CGF-8. (I) CGF-9. (J) CGF-10. (K) CGF-11.
(L) CGF-12. (M) CGF-13. (N) CGF-14. (O) CGF-15. (P) CGF-16. (Q) CGF-17. (R) CGF-18.

A

C

B

Figure 3. Three bacterial colonies used in this study. (A) CGF-1. (B) CGF-6. (C) CGF-11

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Screening results indicated hydrolysis zones produced by CGF-1, CGF-6, and CGF-11, which exhibited
proteolytic indices of 0.2, 0.5, and 0.4 cm, respectively (Table 1). All these three strains demonstrated the ability to inhibit
casein within the medium. The presence of a proteolytic zone signifies the bacterium’s capability to produce protease
enzymes, as this zone results from the bacterial protease activity that cleaves peptide bonds in the casein substrate within
the skim milk agar. Microbial proteases produced extracellularly play a crucial role in protein structural remodeling
through peptide bond hydrolysis. These enzymes exhibit a variety of physicochemical and catalytic properties, including
specific substrate-binding affinities (18). The objective of this test was to assess the bacteria’s ability to produce protease
enzymes, which is essential for identifying potential anticoagulant agents. Protease has extensive functions including as an
anticoagulant. Protease can be categorized into alkaline, serine, and metalloprotease, with alkaline protease being the most
widely used in industrial applications. Protease produced by microorganisms are often more efficient and cost-effective
than those derived from animals and plants, as they can be scaled up, genetically manipulated, and produced consistently
at lower costs (19).
Three proteolytic bacterial isolates (as detailed in Table 1) were obtained from fermented rusip Pacific oyster tissue,
each exhibiting distinct characteristics. Isolates CGF-1 and CGF-6 displayed similar macroscopic traits, while CGF-11
showed distinct differences. Each colony, characterised by unique morphology, was purified through a minimum three
rounds of sub culturing to achieve pure bacterial isolates.
A

B

C

Figure 4. Bacteria that produce protease enzymes show the presence of a proteolytic zone in skim milk agar medium. The
diameter of proteolytic zone: A. CGF-1= 0.2 cm, B. CGF-6= 0.5 cm, C. CGF-11= 0.4 cm.
Table 1. Morphological data of three proteolytic bacteria
Characteristic
Macroscopic
Colony form
Color
Edge
Elevation
Consistency
Microscopic
Form
Color
Gram type
Spore
Proteolytic index

CGF-1

Bacterial Isolate
CGF-6

CGF-11

Punctiform
Transparent
Entire
Convex
Smooth

Punctiform
Transparent
Entire
Convex
Smooth

Irregular
Cream
Undulate
Flat
Smooth

Coccus
Purple
Positive
Negative
0.2cm

Coccus
Purple
Positive
Negative
0.5cm

Bacillus
Red
Negative
Positive
0.4cm

A

B

C

100 nm

100 nm

100 nm

Figure 5. Gram morphology of three proteolytic isolates under the microscopes at 100x magnification.
(A) CGF-1. (B) CGF-6. (C) CGF-011.

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The morphological and biochemical characteristics of the three pure bacterial isolates were identified using Gram
staining and microscopic observation at 100x magnification. The purpose of Gram staining is to classify bacteria into two
groups: Gram-positive and Gram-negative. If the bacteria appear purple, they are classified as Gram-positive; if red, they
are classified as Gram-negative (1). Isolates CGF-1 and CGF-6 were identified as Gram-positive cocci, while isolate CGF-11
was identified as a Gram-negative bacillus (Figure 5). Notably, CGF-1 and CGF-6 formed spores, whereas CGF-11 did not
(Table 1). Spores serve as a defense mechanism for bacteria, enabling them to withstand extreme environmental conditions.
Bacteria that can form spores are often difficult to eradicate because these spores protect them from exposure to
disinfectants (20).

3.3. Molecular Identification
3.3.1. DNA Quantification Test
The basic principle of DNA extraction involves breaking down the cell walls and membranes to isolate the DNA
contained in the nucleoid without damaging it (21). The purity of the isolated DNA was assessed using a NanoDrop™
Spectrophotometer, measuring the absorbance ratio of A260/280. Table 2 presents the purity and concentration levels of
the genomic DNA isolates determined by absorbance analysis. Only isolate CGF-6 was identified in this study, as it exhibited
the highest proteolytic activity, indicating its potential as a source for producing protease enzymes to develop anticoagulant
agents.
DNA is considered pure when the absorbance ratio is 1.8 at λ260/280. Our results indicated a high level of DNA
purity, with an absorbance of 1.96. A ratio between 1.8 and 2.0 signifies high purity. Ratios lower than 1.8 suggest protein
contamination during DNA preparation, while ratios above 2.0 indicate RNA contamination. Such contamination can affect
the accuracy and reliability of molecular analyses (22). Thus, the purity and concentration levels of DNA are critical for the
success of molecular analysis procedures. The A260/280 absorbance ratio is a widely accepted and effective method for
assessing DNA purity and concentration. Nucleic acids absorb maximally at 260 nm, while proteins absorb light more
strongly at 280 nm (23).

3.3.2. Amplification of 16S rRNA Gene
Figure 6 demonstrates the amplification results of the 16s rRNA gene. Genomic DNA extracted from bacterial
isolates served as the template for amplification using the PCR method. The PCR amplification results were subjected to
electrophoresis on a 1% TBE agarose gel and visualized with a UV transilluminator, resulting in a distinct band
approximately 1400 bp in size, as indicated by the DNA marker. Sequencing is conducted after confirming successful
amplification to identify the bacterial species.
The purity and concentration of the extracted DNA significantly affect the visualization of the DNA band. The
amplification of the 16S rRNA gene was performed using the primers Bact 27F and UniB 1492R. The 16S rRNA gene is a
highly conserved region in bacterial genomes, although it undergoes gradual evolutionary changes. Analysing the 16S rRNA
gene is crucial for determining bacterial phylogeny and taxonomy, serving as a universal genetic marker for bacterial
identification. The quality of 16S rRNA gene amplification depends on the size of the amplified DNA fragment (24).
Table 2. Data on the purity and concentration of genetic DNA of the bacterial isolate CGF-6
Isolate
Concentration
A260/280
CGF-6
99.3 ng/µl
1.96

Volume
50 µl

Figure 6. Amplification of CGF-6 DNA using Bact 27F- UniB 1492R primers.
M = 1k b DNA ladder (Cat. No. DL006, Geneaid, Taiwan); 3008 = CGF-6 bacterial isolate

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3.3.3.

Indones J Med Lab Sci Technol. April 2025;7(1):70-79

BLAST Analysis

BLAST analysis refers to http://www.ncbi.nlm.nih.gov. The results of BLAST analysis for the CGF-6 isolate
demonstrated a similarity to species in GenBank (Table 3). The CGF-6 isolate shows a homology value of 99.86% with
Staphylococcus epidermidis. Based on this level of homology, the GGF-6 isolate is classified within the same species as
Staphylococcus epidermidis, belonging to the genus Staphylococcus.
Typically, the homology percentage reflects identity at the genus level but can vary significantly at the species level.
A homology level exceeding 97% generally indicates that two organisms belong to the same species, while levels between
93% and 97% suggests they belong to the same genus. Homology levels below 93% usually indicate family-level differences
(25). However, it is important to note that a homology level below 70% does not necessarily suggest a new species,
especially if there is a lack of data in GenBank, which is insufficient evidence to support such a claim (26).
3.3.4.

Phylogenetic Tree Construction

As reported in Figure 7, it can be indicated that the bacterial isolate CGF-6 is closely related to Staphylococcus
epidermidis strain, as they share a phylogenetic branch, clade, or genus. The same homology level (99.86%) was observed
in various strains, including S. epidermidis strain G003 (accession number KX926554.1), Staphylococcus sp. DMS G06
(accession number KR709224.1), S. capitis subsp. capitis strain Ph 20A1 (accession number KT719989.1), Staphylococcus
sp. strain ZG14-1 (accession number OQ981408.1), S. epidermidis strain SA (accession number OR342081.1), S. epidermidis
strain HDS (accession number OR342084.1), Staphylococcus sp. strain UFLA01-930 (accession number KX555442.1),
Staphylococcus strain SA-144 (accession number KY194740.1), Staphylococcus sp. strain 1910ICU161 (accession number
MT225634.1), and S. epidermidis strain 1910ICU248 (accession number MT225635.1).
Table 3. Top 10 BLAST result
Strain
Species
Staphylococcus
Staphylococcus epidermidis strain G003
epidermidis strain
Staphylococcus sp. strain DMS G06
CGF-6
Staphylococcus capitis subsp. capitis strain Ph20A1
Staphylococcus sp. strain ZG14-1
Staphylococcus epidermidis strain SA
Staphylococcus epidermidis strain HDS
Staphylococcus sp. strain UFLA01-930
Staphylococcus strain
SA-144
Staphylococcus sp. strain 1910ICU161
Staphylococcus epidermidis strain 1910ICU248

Similarity
99.86%
99.86%

Accession Number
KX926554.1
KR709224.1

99.86%

KT719989.1

99.86%
99.86%
99.86%
99.86%

OQ981408.1
OR342081.1
OR342084.1
KX555442.1

99.86%

KY194740.1

99.86%
99.86%

MT225634.1
MT225635.1

CGF-6 Isolate

Figure 7. Phylogenetic tree of CGF-6 bacterial isolates

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S. epidermidis is a Gram-positive, non-spore-forming coccus that belongs to the Staphylococcus genus. It is a common
resident of human mucous membranes, particularly the skin and is recognised as part of the normal human skin microbiota.
Although not inherently pathogenic, some strains exhibit significant halotolerant, allowing them to thrive in marine
environments (27). Numerous studies have explored protease-producing microorganisms within marine biota, including
sea cucumbers, brown algae, shrimp, seaweed, tuna, coral, and nudibranchs. Common isolates from these sources include
bacteria from the genera Staphylococcus, Bacillus, and Pseudomonas (28). Previous studies reported on proteolytic bacteria
isolated from rusip fermented sea cucumber tissue, identified as S. hominis strain HSFT-2 and Bacillus cereus strain HSFI10, which exhibited proteolytic activity (1,28).
Furthermore, Staphylococcus saprophyticus, another protease-producing species of Staphylococcus, has been
successfully isolated from marine sediments. The protease enzyme produced by this strain shows several notable
characteristics, including a molecular weight of 28 kDa, remarkable thermostability (remaining stable at temperatures
ranging from 10 to 80°C), and robust stability against a variety of chemical agents, such as surfactants, oxidizing agents,
bleaching agents, and hydrophobic solvents. These combined properties suggest strong potential for industrial applications
of this enzyme (29).
Protease is an enzyme that catalyses peptide bonds in protein structures. Using appropriate substrates can enhance
the effectiveness of proteases in regulating physiological processes, including fibrinolysis. Fibrinolytic protease belongs to
the serine protease group and is often produced by microorganisms. This type of protease has the same mechanism as
plasmin (protease-like plasmin) in the body and is known to have fibrinolytic activity. Protease like plasmin is a fibrin clotdissolving agent that directly works to dissolve fibrin in blood clots without activating plasminogen like the fibrinolysis
process in the body (30).
Several studies have reported that proteases derived from marine microbes exhibit anticoagulant activity. Bacteria
such as S. hominis, S. aureus, Staphylococcus sciuri, S. saprophyticus, B. cereus, B. thuringiensis, and B. tequilensis demonstrate
anticoagulant properties by prolonging blood clotting times and lysing blood clots in vitro. Notably, novel Staphylokinase
(SAK), a fibrin-specific plasminogen activator, produced by Staphylococcus species, has been identified and shows
established anticoagulant activity. SAK has also been investigated in patients with myocardial infarction (31,32).
In our research, we identified a protease-producing bacterium, specifically the S. epidermidis strain CGF-6. As a
member of the Staphylococcus genus, the strain may possess potential anticoagulant activity that has yet to be investigated.
To assess its potential as an anticoagulant agent, we perform test such as the fibrin plate assay, thrombolytic assay, and
clotting time assay. These tests can help evaluate the anticoagulant activity in vitro by measuring clotting time and the
degradation of fibrin, which are crucial characteristic of effective anticoagulant agents (33).
The protease produced by S. epidermidis CGF-6 belongs to the serine protease family. Serine proteases are found in
both prokaryotic and eukaryotic cells and play roles in cellular and physiological processes, including haemostasis, the
blood clotting cascade, and fibrinolysis. Previous studies have demonstrated the involvement of serine proteases as
anticoagulants and antiplatelets (34).
This study provides valuable insights into potential sources of proteolytic bacteria within rusip Pacific oysters. It
serves as a reference point for identifying protease-producing bacteria with the potential to be developed as anticoagulant
agents. However, it is important to note that this research represents the initial stages of exploring protease-producing
bacteria, and the characterisation and testing of their anticoagulant activity have not yet been addressed. Future research
will involve the extraction of protease enzymes from these bacteria, followed by purification steps utilizing precipitation
and chromatography techniques. Enzyme characterisation will be conducted using zymography methods to gather
comprehensive data on the activity of the protease enzymes.
Evaluating the efficacy of these proteases as anticoagulant agents requires both in vitro and in vivo testing. In vitro
assays will include methodologies, such as fibrinolytic assays, clot formation and lysis assays (CloFAL), Euglobulin clot lysis
assays, anti-platelet aggregation activity assays, and Thromboelastographic. In vivo activity assessments will include the
D-dimer test, ferric chloride-induced thrombosis models, and rat grain flap models (35,36). These assays aim to evaluate
the efficacy and potential pathogenicity of the enzyme candidates as anticoagulant agents. Ultimately, successful outcomes
from these investigations could lead to the development of novel drugs and laboratory diagnostic reagents.

4. CONCLUSIONS
Eighteen bacterial isolates were successfully obtained from the fermentation product of rusip, a traditional dish made
from Pacific oyster. Among these isolates, three were identified as proteolytic bacteria: CGF-1, CGF-6, and CGF-11. Notably,
the CGF-6 isolate demonstrated the highest proteolytic index and was identified as a strain of Staphylococcus epidermidis,
which designated as CGF-6. This isolate shows promise as a source for producing protease enzymes that could be applied
in the development of anticoagulant agents. Further research is needed to purify and characterise these enzymes in order
to optimise their activity. Additionally, in vitro and in vivo studies are essential to evaluate the anticoagulant activity and
efficacy of potential agent candidates.

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Author contributions: MAA: Conceptualization. M, SS: Drafting manuscript. MAA, SSD: Methodology. GDRA: Analysis data
and documentation.
Funding: This research was funded by the Research and Community Service Institute Universitas Muhammadiyah
Semarang, grant number 022/UNIMUS.L/PG/PJ.INT/2023.
Acknowledgements: The author would like to thank the Research and Community Service Universitas Muhammadiyah
Semarang for the support of this research.
Ethics statement: None.
Conflict of interest: The author declares no conflict of interest.

innumerable uses. 3 Biotech. 2021;11(10):1–25.
https://doi.org/10.1007/s13205-021-02928-z

REFERENCES
1.

2.

3.

4.

Fuad H, Hidayati N, Darmawati S, Munandar H,
Sulistyaningtyas
AR,
Ernanto
AR,
et
al.
Exploration of bacteria isolated from “rusip” fermented
tissue of sand sea cucumber holothuria scabra
with fibrinolytic, anticoagulant and antiplatelet
activities.
AACL
Bioflux.
2021;14(3):1242–58.
http://www.bioflux.com.ro/docs/2021.1242-1258.pdf

14. Singh S, Bajaj BK. Potential application spectrum of
microbial proteases for clean and green industrial
production. Energy, Ecol Environ. 2017;2(6):370–86.
https://doi.org/10.1007/s40974-017-0076-5
15. Kumar A, Dhiman S, Krishan B, Samtiya M, Kumari A,
Pathak N, et al. Microbial enzymes and major applications
in the food industry: A concise review. Food Prod Process
Nutr. 2024;6(1):1-6. https://doi.org/10.1186/s43014024-00261-5

Hengkengbala SI, Lintang RA, Sumilat DA, Mangindaan RE,
Ginting
EL,
Tumembouw
S.
Morphological
characteristics and protease enzyme activity of
nudibranch symbiont bacteria. [Karakteristik morfologi
dan aktivitas enzim protease bakteri simbion
nudibranch]. J Pesisir dan Laut Trop. 2021;9(3):83.
https://doi.org/10.35800/jplt.9.3.2021.36672

6.

Ulagesan S, Krishnan S, Nam TJ, Choi YH. A
review of bioactive compounds in oyster shell and
tissues. front Bioeng Biotechnol. 2022;10:1-15.
https://doi.org/10.3389/fbioe.2022.913839

9.

13. Matkawala F, Nighojkar S, Nighojkar A. Next-generation
nutraceuticals:
Bioactive
peptides
from
plant
proteases.
Biotechnologia.
2022;103(4):397–408.
https://doi.org/10.5114/bta.2022.120708

Patantis G, Zilda DS, Li J, Gu X, Gui Y, Ethica SN.
Screening of culturable seaweed associated bacteria
with polysaccharidases activity isolated from the
Ambon Waters, Indonesia. Squalen Bull Mar
Fish
Postharvest
Biotechnol.
2023;18(2):81–92.
https://doi.org/10.15578/squalen.770

Negara BFSP, Mohibbullah M, Sohn JH, Kim JS, Choi JS.
Nutritional value and potential bioactivities of Pacific
oyster (Crassostrea gigas). Int J Food Sci Technol.
2022;57(9):5732–49. https://doi.org/10.1111/ijfs.15939

8.

12. Chapman J, Ismail AE, Dinu CZ. Industrial
applications of enzymes: Recent advances, techniques,
and
outlooks.
Catalysts.
2018;8(6):20–9.
https://doi.org/10.3390/catal8060238

Afriansyah MA, Ethica SN. Fibrinolytic proteaseproducing
bacteria
with
varied
hemolysis
pattern associated with marine algae Dictyota sp.
Med
Lab
Technol
J.
2023;9(2):101–12.
https://doi.org/10.31964/mltj.v9i2.525

5.

7.

11. Ritschard JS, Schuppler M. Cheeses and its impact on
cheese quality and safety. Foods. 2024;13(214):1–33.
https://doi.org/10.3390/foods13020214

16. NOAA FISHERIES. Pacific oyster (Crassostrea gigas)
[Internet].
United
Stated.
2024.
https://www.fisheries.noaa.gov/species/pacific-oyster
17. Kaempe NP, Ethica SN, Sukeksi A, Kartika AI. Isolation
and molecular identification of protease producing
bacterium associated with the brown algae Hydroclathrus
sp. from Hoga Island of Wakatobi District.
Egypt J Aquat Biol Fish. 2024;28(3):637–48.
https://doi.org/10.21608/ejabf.2024.358931
18. Friis L, Heiner C, Bech E, Friis L, García-b B,
Bang-berthelsen CH, et al. Extracellular microbial
proteases with specificity for plant proteins in food
fermentation. Int J Food Microbiol. 2022;381:109889.
https://doi.org/10.1016/j.ijfoodmicro.2022.109889

Lee HJ, Saravana PS, Cho YN, Haq M, Chun BS.
Extraction of bioactive compounds from oyster
(Crassostrea gigas) by pressurized hot water extraction.
J
Supercrit
Fluids.
2018;141:120–7.
Doi:
http://dx.doi.org/10.1016/j.supflu.2018.01.008

19. Ndochinwa OG, Wang QY, Amadi OC, Nwagu TN,
Nnamchi CI, Okeke ES, et al. Current status and emerging
frontiers in enzyme engineering: An industrial
perspective.
Heliyon.
2024;10(11):e32673.
https://doi.org/10.1016/j.heliyon.2024.e32673

Hoang TH, Liang Q, Luo X, Tang Y, Qin JG. Bioactives from
marine animals : Potential benefits for human
reproductive
health.
Frontiers.
2022;9:1–13.
https://doi.org/10.3389/fmars.2022.872775

20. Andryukov BG, Karpenko AA, Lyapun IN. Learning from
nature: Bacterial spores are a target for current medical
technologies. Sovrem Tehnol v Med. 2020;12(3):105–22.
https://doi.org/10.17691/stm2020.12.3.13

Karthikeyan A, Joseph A, Nair BG. Promising bioactive
compounds from the marine environment and their
potential effects on various diseases. J Genet Eng
Biotechnol. 2022; 20(14):1-38. Available from:
https://doi.org/10.1186/s43141-021-00290-4

21. Chang D, Tram K, Li B, Feng Q, Shen Z, Lee CH, et al.
Detection of DNA amplicons of Polymerase Chain Reaction
using
litmus
test.
Sci
Rep.
2017;7(1):1–8.
https://doi.org/10.1038/s41598-017-03009-z

10. Solanki P, Putatunda C, Kumar A, Bhatia R, Walia A.
Microbial proteases: Ubiquitous enzymes with

78

Muhammad Ardi Afriansyah, et al.

Indones J Med Lab Sci Technol. April 2025;7(1):70-79

22. Lucena-Aguilar G, Sánchez-López AM, BarberánAceituno C, Carrillo-Ávila JA, López-Guerrero JA, AguilarQuesada R. DNA source selection for downstream
applications based on DNA quality indicators
analysis. Biopreserv Biobank. 2016;14(4):264–70.
https://doi.org/10.1089/bio.2015.0064

HSFI-10.
Biotropia.
2023;30(2):147–57.
https://doi.org/10.11598/btb.2023.30.2.1765
30. Uttatree S, Charoenpanich J. Purification and
characterization of a harsh conditions-resistant
protease from a new strain of Staphylococcus
saprophyticus. Agric Nat Resour. 2018;52(1):16–23.
https://doi.org/10.1016/j.anres.2018.05.001

23. Lesiani BR, Abror YK, Merdekawati F, Djuminar A. Analysis
of purity and concentration Escherichia coli DNA by boiling
method isolation with addition of proteinase-K and RNase.
Indones J Med Lab Sci Technol. 2023;5(2):160–71.
https://doi.org/10.33086/ijmlst.v5i2.4773

31. Amfar F, Fitri L, Suhartono. Molecular identification of a
new isolate of Actinobacteria atis61 and characterization
of the protease activities. Biodiversitas. 2021;22(3):1564–
9. https://doi.org/10.13057/biodiv/d220363

24. Eren K, Taktakoğlu N, Pirim I. DNA sequencing methods:
From past to present. Eurasian J Med. 2022;54:S47–56.
https://doi.org/10.5152/eurasianjmed.2022.22280

32. Hazare C, Bhagwat P, Singh S, Pillai S. Diverse
origins of fibrinolytic enzymes: A comprehensive
review.
Heliyon.
2024;10(5):1-26.
https://doi.org/10.1016/j.heliyon.2024.e26668

25. Conrad RE, Brink CE, Viver T, Rodriguez-r LM,
Aldeguer-riquelme B, Hatt JK, et al. Microbial species
and intraspecies units exist and are maintained by
ecological cohesiveness coupled to high homologous
recombination. Nature Communications. 2024;1–12.
http://dx.doi.org/10.1038/s41467-024-53787-0

33. Hamdani S, Assitiyani N, Astriany, Singgih M, Ibrahim S.
Isolation and identification of proteolytic bacteria from
pig sludge and protease activity determination Isolation
and identification of proteolytic bacteria from pig
sludge and protease activity determination. IOP Conf.
Series: Earth and Environmental Science. 2019;230:1-8.
https://doi.org/10.1088/1755-1315/230/1/012095

26. Wulandari D, Amatullah LH, Lunggani AT, Pratiwi AR,
Budiharjo A. Antibacterial activity and molecular
identification of soft coral Sinularia sp. symbiont
bacteria from Karimun Jawa Island against skin
pathogens Propionibacterium acnes and Staphylococcus
epidermidis.
BIO
Web
Conf.
2024;92:
1-18.
https://doi.org/10.1051/bioconf/20249202001

34. Sharathkumar HRSMN, Sneharani SDAH. Anticoagulant
and antiplatelet activities of novel serine protease purified
from seeds of Cucumis maderaspatensis. 3 Biotech.
2021;11(1):1–11. https://doi.org/10.1007/s13205-02002565-y

27. Skovdal SM, Jørgensen NP, Meyer RL. JMM
Profile:
Staphylococcus
epidermidis.
J
Med
Microbiol.
2022;71(10):1–5.
https://doi.org/10.1099/jmm.0.001597

35. Nouri K. Mitochondrial ClpP serine proteasebiological function and emerging target for cancer
therapy.
Cell
Death
Dis.
2020;11(10):841;
http://dx.doi.org/10.1038/s41419-020-03062-z

28. Singh R, Gautam P, Sharma C, Osmolovskiy A.
Fibrin and fibrinolytic enzyme cascade in thrombosis:
unravelling
the
role.
Life.
2023;13(11):1-27.
https://doi.org/10.3390/life13112196

36. Krishnamurthy A, Belur PD, Subramanya SB. Methods
available to assess therapeutic potential of fibrinolytic
enzymes of microbial origin: A review. J Anal Sci Technol.
2018;9(1):1-11. https://doi.org/10.1186/s40543-0180143-3

29. Ainutajriani A, Darmawati S, Zilda DS, Afriansyah
MA, Saptaningtyas R, Ethica SN. Production
optimization, partial purification, and thrombolytic
activity evaluation of protease of Bacillus cereus

79