Peptides Hydrolysate Derived from Collagen of Snakehead Murrel (Channa striata) Skin Demonstrate Anti-oxidant and Anti-cholesterol Activities.

PEPTIDES HYDROLYSATE DERIVED FROM COLLAGEN OF
SNAKEHEAD MURREL(Channa striata) SKIN DEMONSTRATE ANTIOXIDANT AND ANTI-CHOLESTEROL ACTIVITIES

WENNY SILVIA LOREN BR SINAGA

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

STATEMENT LETTER OF MANUSCRIPT AND SOURCE OF
INFORMATION*
I declare that this manuscript entitled Peptides hydrolysate derived from
collagen of snakehead murrel (Channa striata) skin demonstrate anti-oxidant
and anti-cholesterol activities is my own work with guidance of the advisors and
has not been submitted in any form at any college, except at Bogor Agricultural
University. Sources of information derived and quoted from published and
unpublished works of other authors mentioned in the text and included in the
reference chapter.
Herewith I bestow the copyright of this manuscript to Bogor Agricultural
University.

Bogor, April 2015
Wenny Silvia Loren Br Sinaga
F251110481

RINGKASAN
WENNY SILVIA LOREN BR SINAGA. Hidrolisat Peptida yang Dihasilkan dari
Kolagen Kulit Ikan Gabus (Channa striata) yang Memiliki Aktivitas Anti-oksidan
dan Anti-kolesterol. Dibimbing oleh MAGGY T. SUHARTONO dan
RAYMOND R. TJANDRAWINATA.
Anti-kolesterol dan anti-oksidan memiliki peranan penting untuk
menghambat penyakit kardiovaskular, terkait dengan adanya gangguan pada arteri
yang disebabkan oleh kolesterol yang teroksidasi. Beberapa tahun belakangan ini
sejumlah riset peptida bioaktif memperlihatkan aktivitas sebagai anti-kolesterol
dan anti-oksidan. Pada penelitian ini, Acid Soluble Collagen di ekstrak dari kulit
ikan gabus dan digunakan sebagai inducer untuk menghasilkan kolagenase oleh
Bacillus licheniformis F11.4.
Kolagenase yang dihasilkan di murnikan menggunakan AKTA Purifier (ion
exchange, DEAE column) dan mendapatkan fraksi D dan F. Fraksi enzim D dan F
kemudian digunakan untuk membuat hidrolisat peptida dari acid soluble collagen.
Hidrolisat peptida yang dihasilkan fraksi D menunjukkan aktivitas inhibitor

HMG-CoA sebanding dengan pravastatin dan sedikit aktivitas antioksidan.
Sementara, hidrolisat peptide oleh F memiliki aktivitas antioksidan yang lebih
sedikit dibandingkan dengan BHT (2mM), vitamin C (2mM) and vitamin E
(2mM), tetapi sedikit aktivitas penghambatan HMG-CoA.
Kata

Kunci:

Anti-cholesterol, anti-oxidant,
collagenase, snakehead murrel

bioactive

peptide,

collagen,

SUMMARY
WENNY SILVIA LOREN BR SINAGA. Peptides Hydrolysate Derived from
Collagen of Snakehead Murrel (Channa striata) Skin Demonstrate Anti-oxidant

and Anti-cholesterol Activities. Supervised by MAGGY T. SUHARTONO dan
RAYMOND R. TJANDRAWINATA.
Anti-cholesterol and anti-oxidant play crucial role to combat cardiovascular
disease (CVD) related to formation of arterial plagues from oxidation of
cholesterol. In the past decades, research on bioactive peptides demonstrating
anti-cholesterol and anti-oxidant activities have been reported as the alternative
drugs. In this study, acid soluble collagen was extracted from the skin of
snakehead murrel and employed to induce production of collagenase by Bacillus
licheniformis F11.4. The collagenases secreted were in turn purified through
AKTA Purifier (ion exchange, DEAE column) and used to produce peptides
hydrolysate.
The purified enzymes were grouped in two distinct collagenase fractions,
designated as fraction D and F. Peptides hydrolysate produced by the fraction D
was found to demonstrate HMG-CoA inhibitor activity comparable to pravastatin
and limited anti-oxidant activity. Meanwhile, peptides hydrolysate generated
using the fraction F demonstrated anti-oxidant activity comparable to BHT
(2mM), vitamin C (2mM) and vitamin E (2mM), but limited HMG-CoA activity.
Combination of the fraction D and F resulted in substantial HMG-CoA inhibition
and anti-oxidant activities.
Keyword: Anti-cholesterol, anti-oxidant, bioactive peptide, collagen, collagenase,

snakehead murrel

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PEPTIDES HYDROLYSATE DERIVED FROM
COLLAGEN OF SNAKEHEAD MURREL (Channa striata)
SKIN DEMONSTRATE ANTI-OXIDANT AND ANTICHOLESTEROL ACTIVITIES

WENNY SILVIA LOREN BR SINAGA

Thesis
As a requirement to obtain
Magister Science degree

in
Food Science Study Program

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

External Thesis Examiner:

Puspo Edi Giriwono, PhD

PREFACE
Praise to the God almighty for thy mercy, blessing, and guidance through
out the research and finishing this manuscript. The research entitled “Peptides
hydrolysate derived from collagen of snakehead murrel (Channa striata) skin
demonstrate anti-oxidant and anti-cholesterol activities” was carried out in Bogor
Agricultural University and DLBS from January 2013 until February 2014.
By completion of this research and manuscript, the author would like to
express great appreciation and sincere thanks to:

1. Prof. Dr. Ir. Maggy T Suhartono, as my supervisor, for her guidance, spirit,
help, and advices to see the world during completing this research and
manuscript.
2. Raymond R. Tjandrawinata, PhD, MS, MBA as co supervisor for his time,
help, advice and opportunity to finish my research in DLBS.
3. Dr. Wangsa Tirta Ismaya as expert trainer in DLBS, for his correction in this
thesis, his comment, his help during the research, and time answering my
question.
4. Dr. Puspo Edi Giriwono as examiner, for his correction, help and comment.
5. My parents (Robinson Sinaga and Rehulina Br. Simbolon)and broher (Zohdy
Gokta Sinaga) for their Prayer, Believing and support during the study of Food
Science and Technology, the research, and manuscript completion.
6. Dexa Laboratories for Biomoleculer Science for the time and chance to do my
research was. Ibu Debbie S. Retnoningrum who held responsible in incentive
Sinas funding and also for the DLBS members (Bu Henny, Pak Bambang, Mba
Hayu, Ka Lolen, Ka Frans, Mas Yogi, Kak Lia, Apry, Bang Madan, Apryani
Rahma, Utha, Tia, Putri, Pak Dokter, Mba Aini dan Mba Chandra) For their
guidance and help in the laboratory.
7. Food science student (Rahma, Mba Diana Nur Afifah, Silvie, Novan, mba Ino,
Diana Lestari, Ibu Wati, Adi, Rahayu, Risma, Nisa, Reny, Septi otong, Ella,

Pak Rinto, Ka Becky, Ayu, Evelyn and others) and for Bu Ika for their support,
valuable friendship, and unforgettable moments during the study and
completion of this research.
8. Special Thanks to my Cikuci (DJ Calvien H) for his prayer, believing,
loquaciousness, advice, patient, time, support, lend a hand and shoulder when I
cry and need a help and guidance during the study and finish this thesis.
9. GSP IPB and Psaltrio singer (Devide, Silvia, Ka Jems, Arif, Nas, Merry,
Yuang, Sars, Roto, Mas Deka, Pak John, Ka Elga, Ka Tasha & Bang Anton,
Mba Rini, Ka Tere, Kinan and Mas Ari) with their happiness shared.
10. PP YN Crew (Dessy Cenion and Minion, Btari Sisters, Situmorang Sisters,
Monce, Erti, Risma, Risty, Kristin, Mele and the ganks, Risvan, Dira and
Others) for their support, long lasting friendship, waiting me when insomnia
catch me and precious moments shared during this manuscript written.
Last but not least, hopefully this manuscript is useful for the readers and gives
a contribution in food science development.
Bogor, August 2015

Wenny Silvia Loren Br Sinaga

CONTENT

LIST OF FIGURE

vi

LIST OF APPENDIX

vi

1 INTRODUCTION

1

Background

1

Problem Statement
Objective
Significance of study

2
2
2

2 LITERATURE REVIEW

2

3 RESEARCH METHODOLOGY
Organisms and materials
Production and partial purification of collagenases
Collagenase assay and protein concentration determination
SDS PAGE and zymogram
Preparation of bioactive peptides hydrolysate
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay
HMG-CoA reductase assay

5
5
5

6
6
6
7
7

4 RESULT AND DISCUSSION
Partial purification and characterization of collagenases
Anti-oxidant activity
Anti-cholesterol activity

7
7
12
12

5 CONCLUSSION AND RECOMMENDATION
Conclusion
Recommendation
Acknowledgements

14
14
14
14

REFFERENCE

14

AUTHOR BIOGRAPHY

24

APPENDIX

19

BIOGRAPHY

24

LIST OF FIGURE
Figure 1 (A) Graphic from AKTA Purifier IEC(DEAE) by using linear
gradient salt elution (sample injection: 2mL, 45 CV, Flow rate:
5ml/mnt, Elution: 5ml). (B) Zymography analysis of the
enzymes after purification. C: crude enzyme, As: after
ammonium sulphate fractionation, 8-50: fractions of enzyme
Figure 2 Zymography analysis of enzyme from purification upon
challenge with proteases inhibitors. C: crude enzyme, As: after
ammonium sulphate fractionation, and 8-50: fractions of
enzyme. Fractions 26-34 are D whilst fractions 40 and 50 are F.
Notation E and P refers to EDTA and PMSF, respectively.
Figure 3 (A). Graphic from AKTA Purifier by using stepwise salt elution
(sample injection: 65mL, 40 CV, Flow rate: 0.8ml/mnt, Elution:
2ml). (B).Zymography analysis of the enzymes after
purification on anion exchanger column. C: crude enzyme, As:
after ammonium sulphate fractionation, 18-95: fractions of
enzymes.
Figure 4 (A). Graphic from AKTA Purifier by using stepwise elution
from first purification from collected fraction 48-80 (Figure
3A), (sample injection: 2mL, 45 CV, Flow rate: 0.8ml/mnt,
Elution: 2ml). (B).Zymography analysis of the enzymes after
purification on anion exchanger column. As: after ammonium
sulphate fractionation, 17-78: fractions of enzymes.
Figure 5 Antioxidant Activity
Figure 6 Inhibition of HMG-CoA reductase

8

9

10

11
12
13

LIST OF APPENDIX
Appendix1 Reagents for Bradford Assay
Appendix2 Composition of standard solution for Bradford assay
Appendix4 Standard Curve of Bovine Serum Albumine (BSA)
Appendix5 Reagents for SDS-PAGE analysis
Appendix6 Composition of separating and stacking gels for SDS-PAGE
and Zymography analysis

19
20
21
22
23

1 INTRODUCTION
Background
Bioactive peptide (BP) is derived from proteins through acid or proteolytic
activity (Senevirathne and Kim, 2012). BP has been developed into antihypertension, anti-oxidant, anti-thrombotic, and hypo-cholesterolemic drugs,
which are potent to prevent degenerative diseases, such as cardiovascular disease
(CVD). Protein for the source of BP can be from plants, meat, milk (Korhonen
and Pihlanto, 2003; Korhonen and Pihlanto, 2006 ) and fish (Senevirathne and
Kim, 2012). An example of anti-oxidative BP is lunasin, which is identified in
soybean and other plants (Galvez and Lumen, 1999). This peptide is already
commercialized in the US and reported to decrease low density lipoprotein (LDL)
and cholesterol in the blood (Galvez, 2012).
Recent studies have shown that anti-oxidative BP can be released from
casein through enzymatic hydrolysis or during fermentation of milk using
protease-producing lactic acid bacteria (Korhonen and Pihlanto, 2003). The antioxidative BP displays free radical scavenging activities and inhibits enzymatic
and non-enzymatic lipid peroxidation, most likely for being a preferred target over
fatty acid free radicals (Rival et al., 2001). Consumption of anti-oxidative BP
derived from goat is reported to show anti-atherogenicity by prolonging resistance
of the lipoprotein fraction to oxidation (Kullisaar et al., 2003).
Snakehead murrel is one of the freshwater fishes from Channidae family
with high collagen content in their skin. The skin of fish has been considered as
waste in the fish processing. In this study, collagen was extracted from fish skin
by acid treatment and found that the peptides hydrolysate derived from collagen
was able to inhibit the activity of HMG-CoA reductase, the key enzyme for
cholesterol biosynthesis. Anti-oxidant activities are detected in this peptides
hydrolysate. The peptides were produced by proteolytic digestion using
collagenases from B. licheniformis F11.4, a mutant of B. licheniformis from
Indonesia. This bacterium has previously been shown to demonstrate high
proteolytic and collagenolytic activities (Waldek et al., 2006), secreting
collagenases of 124 kDa and 26 kDa when grown in the presence of water-soluble
collagen derived from milkfish skin (Baehaki et al., 2012; Baehaki et al., 2014).
Our present study revealed the potential use of the peptides hydrolysate
derived from the snakehead murrel skin acid soluble collagen as anti-cholesterol
and anti-oxidant agents. This is the first report on the use of bioactive peptides
hydrolysate derived from collagen of snakehead skin. Also, while most marine
bioactive peptides are developed as anti-hypertension agent, this report explored
the possibility of application of marine bioactive peptides as anti-cholesterol and
anti-oxidant instead. This report paves foundation for further identification of the
bioactive peptides and structural-function study of the enzymes.

2
Problem Statement
Bioactive peptide is known to have anti-cholesterol and anti-oxidant
activities. Non-enzymatic method to make bioactive peptide can produce
unspecific peptides while enzymatic protein degradation can work specifically to
degrade protein more effectively. In previous study, Bacillus licheniformis F11.4
was found to produce collagenase. Purified enzyme is needed to apply and find
the good characteristics of this peptides hydrolysate. In this study Snakehead
murrel (Channa striata) skin was extracted by using Acid Solluble Collagen
(ASC) method and used it as inducer to get collagenase from Bacillus
licheniformis F11.4 and applied the enzyme to promote peptides hydrolysate and
analyze the bioactivity.
Objective
The aim of this research was: 1) to purify collagenase from B. licheniformis
F11.4 and apply the enzyme for production of collagen bioactive peptide
hydrolysate. 2) to characterize peptides hydrolysate specifically their anticholesterol and anti-oxidant activities.
Significance of study
This study paves foundation for further enzyme purification and
identification of the bioactive peptides and structural-function study of the
enzymes.

2 LITERATURE REVIEW
Several proteases from microorganisms have been reported. Collagenaseproducing Bacillus reported include: Bacillus licheniformis N22 (Asdornnithee et.
al. 1994), Bacillus subtilis FS-2 (Nagano and To 1999), B. subtilis CN2 (Tran and
Nagano 2002), Bacillus sp. MO-1 (Okamoto et. al. 2001), B. subtilis AS1.398
(Riu et. al. 2009), Bacillus pumilus CoI-J (Wu e.t al. 2010), Streptomyces sp.
Strain 3B (Petrova et. al. 2006) and Streptomyces parvulus (Sakurai et. al. 2009).
Serine collagenase is often characterized with their molecular weight of 24-36
kDa (Roy et. al. 1996), while metallo-collagenase is 30 to 150 kDa (Harris and
Vatar 1982). Some metallo-collagenases contain zinc and require calcium for
stability (Stricklin et. al. 1977).
B. lichenifromis F11 show high protease (collagenase) activity but lack
chitinase activities. B. licheniformis F11.4 displayed rough colony morphology.
Microscopic investigation revealed form motile rods of equal sizes (2.9 by 0.75
μm). According to the physiological and microscopic tests, B. licheniformis F11 is
suggested to be representatives of the B. licheniformis species (Waldeck et. al.
2006). These findings agree with the fact that no extra-chromosomal elements
(approximately 1 to 20 kb in size) could be detected within F11cells since small
plasmids are rarely found in B. licheniformis strains (6 to 25%). However, definite
evidence for the affiliation of the isolates with the species B. licheniformis was

3
obtained from sequencing the 16S rRNA gene, including the hypervariable
regions V1 to V3; 100% identity to B. licheniformis DSM13/ATCC 14580, which
was totally sequenced only recently, was found (Waldeck et. al. 2006). By
targeted deletion of the polyglutamate operon (pga) in B. licheniformis F11, a
derivative form, F11.1 (pga), was obtained, along with lacking polyglutamate
(PGA) formation and, enhanced proteolytic activities. The phenotypic properties
were maintained in a strain in which the chiBA operon was additionally deleted:
F11.4 (chiBA pga) (Hoffmann et. al. 2010).
Collagen is partly responsible for toughness in red meat and used as
tenderizers in the food industry (Cronlund and Woychik 1987). Collagenases have
applications in fur and hide tanning to help ensure a uniform dying of leathers
(Goshev et. al. 2005; Kanth et. al. 2008). However, the most common uses of
these enzymes appear to be in medicine. They are used to treat burns and ulcers
(Agren et. al. 1992), to eliminate scar tissue (Shmoilov et. al. 2006) and play an
important role in the successful transplantation of specific organs (Klock et. al.
1996; Kin et. al. 2007). Chung et. al. (2004) found catalytic domain which have
Zn in their active site bind with collagen. Collagenase will loose a rigid triplehelix of collagen. Bond of Gly 775 – Ile 776 amino acid were cleave into the typical
three-quarter and one-quarter fragments.
Bioactive peptides are proteins synthesized in the cell in the form of large
prepropeptides, which are then cleaved and modified to give active products. Milk
proteins are a rich source of biologically active peptides such as antihypertensive,
antithrombotic, immune-stimulating, antimicrobial, mineral carrying and
cholesterol lowering-peptides (Shah 2000). Many researchers are interested to
solve the question about the importance of bioactive foods are as food constituents
or as drugs and it needs careful examination. ACE inhibitory peptides,
immunomodulating peptides, and caseinophosphophopeptides are the most
favorite bioactive peptides for application to food stuffs formulated to provide
specific health benefits. Casein derived peptides have already found interesting
applications as dietary supplements and as pharmaceutical preparations such as
tablets, toothpaste, and dental filling material. The efficacy and safe conditions of
use of these peptides in animals and in humans remain yet to be proven.
The importance of oxidation in the body and in food stuffs has been widely
recognized. Oxidative metabolism is essential for survival of cells. A side effect
of this dependence is the production of free radicals and other reactive oxygen
species that cause oxidative changes. When an excess of free radicals is formed,
they can overwhelm protective enzymes like superoxide dismutase, catalase and
peroxidase which cause destructive and lethal cellular effects (e.g. apoptosis) by
oxidizing membrane lipids, cellular proteins, DNA, and enzymes thus shutting
down cellular process. Recent studies have shown that anti-oxidative peptides can
be released from caseins in hydrolysis by digestive enzymes and in fermentation
of milk with proteolytic lactic acid bacteria strains (Korhonen and Pihlanto, 2003).
Most of the identified peptides are derived from αs-casein and have been
shown to possess free radical scavenging activities and to inhibit enzymatic and
non enzymatic lipid peroxidation, most likely by being a preferred target over
fatty acid free radicals. The consumption of fermented goat milk improved
antiatherogenicity in healthy subjects by prolonging the resistance of the
lipoprotein fraction to oxidation, lowering the levels of peroxidized lipoproteins,

4
oxidized LDL, 8-isoprostanes and the glutathione redox reaction, and enhancing
total antioxidative activity. Therefore, it is hypothesized that low antioxidant
levels may increase coronary heart disease. More research is needed to elucidate
the role of antioxidative peptides in the protective functions in human. Not only
from fermented milk, some research which have antioxidant activity have been
reported such as soy protein, pig meat protein, seeds protein, and some fish.
The 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase
inhibitors or statins, are potent inhibitors of cholesterol biosynthesis that are
extensively used in the treatment of patients with hypercholesterolemia. Statins
impair cholesterol synthesis by inhibiting the activity of the enzyme HMG-CoA
reductase. Inhibiton of cholesterol biosynthesis is accompanied by an increase in
low-density lipoprotein (LDL) receptors in the liver, leading to increase uptake
and clearance of cholesterol from plasma (Borghi et. al. 2002).
Once a crude collagenase extract is recovered, it can be purified by their
physicochemical properties such as ion charge, size exclusion, hydrophobic
interaction or affinity. ion-exchange chromatography will separate ions and polar
molecules according to charge of protein. Separation can be achieved based on the
natural isoelectric point of the protein. At a given pH most proteins have an
overall negative or positive charge depending on their isoelectric point (pI), hence
these proteins interact with an oppositely charged resin packed in a column.
Proteins are retained according to their charge. There are two commonly used
types of columns: (a) Sephadex Diethylaminoethyl (DEAE) cellulose for binding
to negative charge of proteins and (b) Carboxymethyl (CM) for binding to net
positively charged proteins (Kaufman et. al. 1995). At optimal pH levels (near
7.0), most of collagenases bear an overall negative charge and anionic exchange
column should be employed. Anion exchange chromatography based on
Diethylaminoethyl (DEAE) cellulose or agarose is by far the most common ion
exchange technique that has been used by a number of researchers to partially
purify collagenase (Kim et. al. 2002).
Gel filtration is known as size exclusion or molecular sieve
chromatography. It separates molecules based on size. It is a physical separation
where the column packing material contains pores that only molecules within a
particular size or mass range can enter and be retained. Many commercial gel
matrixes are used such as Sephadex (10, 25, 50, 75, 100 and G-200), Sepharose
(6B, 4B, 2B), Sephacryl (S-200, S-300, S-400), and Bio-Gel (P-10, P-30, P-60, P100, P-150, P-200, P-300, A 0.5-50). These different materials have different
protein size-exclusion ranges. Proteins can be separated by passing them through
the appropriate column. Several researchers performed collagenase purification
using gel-filtration chromatography. Ohyama and Hashimoto (1977) used
Sephadex G-150 to purify collagenase of human skin. Indra et. al. (2005) used
gel-filtration chromatography on Sephadex G-100 to obtain purified collagenase
from hepatopancreas of the marine crab and land snail, respectively. Several gelpowders have been used including: Polyacrylamide and mixtures of
polyacrylamide and dextran (Sakurai et. al. 2009). Regardless of support material,
all groups seem to consistently employ a maximum pore size such that 200 kDa is
the upper molecular mass limit for solute retention (i.e., all molecules with large
molecular masses pass through the column with the solvent).

5
HIC makes use of surface hydrophobicity interaction of protein and column
packing material in the presence of salt concentrations. Because amino acids have
different hydrophobicity, HIC can be used to separate proteins and enzymes with
different compositions. This technique is similar to Reverse Phase (RP)
chromatography (HPLC application) where non-polar structures, such as alkyl
ligands, are attached to a support and form reversible interactions with the solutes.
With HIC, however, the interactions are much weaker so that proteins are not
strongly retained and can be recovered with polar solvents with varying salt
concentrations (Queiroz et. al. 2001). Kristjansson et. al. (1995) used a column of
phenyl-substituted agarose to partially purify collagenase. Sakurai et. al. (2009)
used this technique but with a more hydrophobic butyl substitute.
Affinity chromatography uses a specific interaction between a substrate and
a biologically active substance and is the most powerful method for protein
purification. This method is based on the high affinity of some proteins to specific
chemical groups (ligands) covalently attached to a chromatographic column
packing material (Kaufman et. al. 1995). The interaction may be very specific,
with a substrate bonded to the stationary phase that only the enzyme of interest
can interact with such as a collagen-collagenase pairing. Stationary phases with
collagen as the ligand are not commercially available but a number of research
groups have prepared their own packing materials to make use of this very
specific interaction (Tyagi and Cleutjens 1996). More general interactions involve
a ligand bound to the stationary phase that interacts with a number of molecules
that contain certain structures.

3 RESEARCH METHODOLOGY
Organisms and materials
Bacillus licheniformis F11.4 was kindly provided by the Indonesian
government agency for assessment and application of technology (BPPT). The
bacterial mutant is derived from B. licheniformis F.11, which was discovered
during research collaboration between BPPT and Munster University - Germany
under the Indo-German Biotechnology scheme. Collagen from snakehead fish
skin was prepared according to Singh et. al. (2011). Chemicals were purchased
from Merck, Sigma, or Oxoid, through local distributors, except when specifically
mentioned.
Production and partial purification of collagenases
B. licheniformis F11.4 was grown in a medium containing 1% NaCl, 0.5%
tryptone, 0.25% yeast extract, and 5% collagen, for 24 hours at 37°C with
agitation speed of 120 rpm. Collagenase secreted into the medium was harvested
by cold centrifugation (4°C) for 15 minutes at 7500 g prior to ammonium sulphate
precipitation at 50% saturation (w/v). The protein precipitate was dissolved in
0.02 M phosphate buffer, pH 8.0, and dialyzed (MWCO 10 kDa) overnight
against 0.01 M phosphate buffer, pH 8.0, at 4°C. The dialyzed sample, designated
as the crude enzyme, was applied to DEAE-Sepharose anion exchanger column

6
(GE Healthcare Life Science, Pittsburg – PA, USA) that had previously been
equilibrated with 0.02 M phosphate buffer, pH 8.0. The column was eluted with a
gradient of 0-1M NaCl in 0.02 M phosphate buffer, pH 8.0, at a flow rate of 0.5
ml/min. The purification was performed on Äkta purifier system (GE Healthcare
Life Science, Pittsburg – PA, USA) at room temperature (25 oC).
Collagenase assay and protein concentration determination
Protease activity was measured using 5% collagen as the substrate
(Bergmeyer et. al. 1983). Briefly, 50μl of enzyme was mixed with 250 μl of
substrate. The mixture was incubated for ten minutes at 37°C. The enzymatic
hydrolysis was stopped by addition of 1250 μl of 0.2 M trichloroacetic acid
(TCA), followed by cold centrifugation at 4,000 g for ten minutes. The
supernatant was mixed with 0.4 M Na2CO3, followed by addition of Follin reagent
at a ratio of 1:2. The mixture was further incubated at 37°C for 20 minutes. The
amount of amino acid produced was measured at 578 nm. One unit of enzyme
activity (U) is defined as the amount of enzyme required to produce 1 μmol of
amino acid per minute under specific conditions. Protein concentration was
determined according to Bradford (1976) using bovine serum albumin (BSA) as
the standard.
SDS PAGE and zymogram
Molecular weight of proteins was estimated using SDS-PAGE (Laemmli
1970). 80 μL samples were mixed with 20 μL buffers (contained Tris-HCl,
glycerol, SDS, β-mercaptoethanol, and bromphenol blue) and it was heated in
boiling water for 5 minutes. The samples were injected into the gel which
contained 12% acrylamide for the separating gel and 4% acrylamide for the
stacking gel (Appendix 5). The electrophoresis process was run at 70 Volt and 50
mA for 2.15-2.31 hours. After that, the gel was stained in staining solution for
several hours and it was destained with destaining solution until the blue bands
were contrast with the gel. Composition of reagents for SDS-PAGE analysis can
be seen in (Appendix 6). High molecular weight markers were used to estimate
the molecular weight of protein.
Enzymes activity in situ was demonstrated in a zymogram gel (Choi et. al.
2000), using 10% non-denaturing PA gel containing 0.1% (w/v) collagen. The
methodology was same as SDS PAGE. The differentiation was the acrilamide
contain and the samples were not boiled in water.
Preparation of bioactive peptides hydrolysate
One ml of partially purified enzymes (0.4-0.7 mg/ml) was added into 50 ml
collagen solution (0.5-1 mg/ml) and mixed with 40 ml phosphate buffer 500 mM.
The mixture was incubated for 120 minutes at 40oC. The enzymatic hydrolysis
was stopped by an addition of 250 mM TCA. The peptide solution was recovered
after centrifugation at 4,000 g in 4ºC and stored at -20oC. For inhibition studies,
similar reaction was conducted at small scale (one tenth) in the presence of 20 µ l
of 5 mM ethylene diamine tetra acetatic acid (EDTA) or phenyl methane sulfonyl
fluoride (PMSF).

7
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay
The anti-oxidant measurement was performed according to Li et. al. (2007).
Briefly, 500 µl of peptide solution was mixed with 500 µL of 99.5% ethanol and
125µL 0.02% DPPH in 99.5% ethanol. The mixture was kept in the dark for one
hour at room temperature. Degradation of DPPH was measured at 517 nm.
Radical scavenging activity (RSA) was calculated as a percentage of the activity
of the control (absence of antioxidant).

HMG-CoA reductase assay
HMG-CoA reductase assay was performed according to Perchellet et. al.
(2009). Prior to the assay, a spectrophotometer was conditioned at 37°C and the
measurement was carried out at 340 nm with and interval reading of 15 seconds
for five minutes. The reaction mixture contained 5 μl Pravastatin or peptides
hydrolysate (the assay buffer as the blank), NADPH, HMG-CoA substrate, and
HMG-CoA Reductase (HMGR) (the assay buffer for the negative control and
blank). The reaction mixture was thoroughly mixed prior to measurement in the
spectrometer. The specific activity of enzyme was calculated according to the
following equation:

Where:
12.44 = εmM - the extinction coefficient for NADPH at 340 nm is 6.22 mM–1cm–1.
12.44 represents the 2 NADPH consumed in the reaction.
TV = Total volume of the reaction in ml (ml)
V
= Volume of enzyme used (ml)
0.6 = Enzyme concentration (mg/ml)
LP = Light path in cm (1 cm)
One unit of enzyme is defined as the amount of enzyme required to convert
one milli mole of NADPH to NADP+ per minute at 37°C.

4 RESULT AND DISCUSSION
Partial purification and characterization of collagenases
Snakehead fish (Channa striata) has been exploited for application in
protein therapy, such as wound healing (Baie and Sheikh, 2000). The skin of the
fish contains high amount of proteins in a form of collagen (17% w/w) that can be
used, for example, to prepare gelatin (See et al., 2010). The skin part is mostly
discarded after the fish processing (Shahidi, 1995), thus employing it as the
collagen for generation of bioactive peptides is advantageous.

8
(A)

(B)

Figure 1 (A) Graphic from AKTA Purifier IEC(DEAE) by using linear gradient
salt elution (sample injection: 2mL, 45 CV, Flow rate: 5ml/mnt,
Elution: 5ml). (B) Zymography analysis of the enzymes after
purification. C: crude enzyme, As: after ammonium sulphate
fractionation, 8-50: fractions of enzymes.
Reason for the popularity of IEC (ion-exchange chromatography) include its
(1) high resolving power, (2) high protein-binding capacity, (3) versatility (there
are several types of ion exchangers, and the composition of the buffer and pH can
be varied over a mile range), (4) straightforward separation principle (primarily
according to differences in charge), and (5) ease of performance. First fractions
collected during linear elution of DEAE anion exchanger column with NaCl at a
gradient concentration of 0-1 M, showed the presence of at least seven
collagenases (b1-b7) secreted by B. licheniformis F11.4 (Figure 1B). This first
initiation method used to find the best column for our protease. The result was
DEAE is the best column to separate our enzyme (collagenase). The collagenases
were classified by means of protease inhibitors such as, PMSF (Phenyl methyl
sulfonyl fluoride) for serine protease and EDTA (Ethylene diamine tetra acetic

9
acid) for metallo-protease (Figure 2). Interestingly, the inhibition profile identified
b1 as serine protease, b2 as metallo-protease, and neither of both (b3-b7). The
zymography assay may indicate the presence of possible dimer, trimer, or other
oligomeric enzyme states, which are undetectable in SDS PAGE under denaturing
conditions. Further, b7 was discovered to actually contain likely two species,
which are neither serine nor metallo-protease (27) and a serine protease (40, and
50). The b7 enzyme (Figure 1B) was probably a very small enzyme or an enzyme
fragment that still contains the active site of collagenase. Unfortunately, this
molecular information cannot yet be disclosed. Nevertheless, the characterization
of partially purified enzyme indicated the presence of more than one collagenase
with different characteristics. Namely, a serine protease inhibited by PMSF and a
metallo-protease inhibited by EDTA.

Figure 2 Zymography analysis of enzyme from purification upon challenge
with proteases inhibitors. C: crude enzyme, As: after ammonium
sulphate fractionation, and 8-50: fractions of enzyme. Fractions 2634 are D whilst fractions 40 and 50 are F. Notation E and P refers to
EDTA and PMSF, respectively.
The enzyme mixture from the first purification using Ion Exchange
Chromatography (DEAE 5ml) with NaCl (0-1M) gradient elution (Figure 1A) was
not separated clearly. Another method to get better separation of enzyme fraction
is stepwise salt elution (0%, 25%, 35%, 60%, 100% of NaCl (1M)) and 8CV
(Column Volume) each step (Figure 3A). Direct sample injection was applied to
the column. Around 65mL of protein samples were injected to get the high
activity of collagenase fraction.
Figure 3 showed that at 35% of salt concentration collagenase fraction 40
and 50 appeared as different protein. But colagenase fraction 50 and 69 (at 60%
salt concentration) were similar. This result implies that the percentage of salt
needed to be within smaller range. The collagenases from the first stepwise
purification (fraction 49 to 80) were pooled to purify in the second stepwise
purification. Before purifying the enzyme, the samples were centrifuged around 5
minutes and 5000rpm until 2mL of collagenase was obtained. Collagenase from
the first stepwise purification was purified again to second purification to get the
best purified collagenase. Figure 4 shows that the collagenase has been well
separated by using stepwise salt elution (0%, 25%, 30%, 40%, 50%, 60%, and

10
100% of NaCl). At the 40% salt concentration (Figure 4B) the collagenase was
well separated. Maybe at this concentration salt can elute better the collagenase.
Collagenase b7 (fraction 73 in Figure 3B and fraction 61 in Figure 4B) were
separate from the b1-b6 fractions.
(A)

(B)

Figure 3 (A). Graphic from AKTA Purifier by using stepwise salt elution
(sample injection: 65mL, 40 CV, Flow rate: 0.8ml/mnt, Elution:
2ml). (B).Zymography analysis of the enzymes after purification on
anion exchanger column. C: crude enzyme, As: after ammonium
sulphate fractionation, 18-95: fractions of enzymes.
The latter fractions 37-41was designated as fraction D while fractions 61-78
were combined and designated as fraction F (Figure 4B). Henceforth, the
collagenase samples used were addressed as the fraction D and F. Peptides
hydrolysate from snakehead fish skin collagen was generated through enzymatic
hydrolysis using collagenases from B. licheniformis F11.4, a mutant of B.
licheniformis 11 from Indonesia (Waldeck et al., 2006). Although SDS PAGE and
zymography analysis were unable to clearly classify all collagenase fractions
secreted, the inhibition study clearly shown the presence of serine-, metallo-, and
neither of both types of proteases. The previous study using collagen from the
skin of milkfish (Chanos chanos) indicated secretion of only two metallo-

11
collagenases with apparent molecular weight of 26 kDa and 124 kDa (Baehaki et
al., 2012; Baehaki et al., 2014). This study could not yet exclude the presence of
various oligomeric or even partial degradation forms of the enzymes. This issue
would be solved in a peptide-mass finger printing analysis, which would be the
(A)

(B)

Figure 4 (A). Graphic from AKTA Purifier by using stepwise elution from first
purification from collected fraction 48-80 (Figure 3A), (sample
injection: 2mL, 45 CV, Flow rate: 0.8ml/mnt, Elution: 2ml).
(B).Zymography analysis of the enzymes after purification on anion
exchanger column. As: after ammonium sulphate fractionation, 17-78:
fractions of enzymes.
next experiments to do. In particular, species b3 to b6 demonstrated similar
bioactivity during the collagenase assay in situ, during which all were identified as
neither serine nor metallo-protease. This finding is interesting because most of
collagenases from Bacillus are characterized as metallo-protease (Baehaki et al.,
2012; Baehaki et al., 2014; Liu et al., 2010), serine protease (Nagano and To,
1999), or Ca2+-dependent and with disulfide bonds collagenase (Wu et al., 2009).
Also, the finding of species F is very interesting because the enzyme is
significantly smaller than the reported collagenases from other strains of Bacillus,

12
e.g. from B. cereus MBL13 (~38 kDa), B. circulans (~39 kDa), B. cereus Soc67
(~88kDa), B. cereus non-haemolytic enterotoxin (Nhe) (~105 kDa), and B.
substilis FS-2 (~125 kDa) (Liu et al., 2010; Lund and Granum, 1999; Maäkinen
and Maäkinen, 1987; Nagano and To, 1999;, Rao et al., 2009). This small enzyme
species could have been a product of autolysis of the larger collagenase and still
contains the active site (Vasilyeva, 2002). Similar situation has been reported for
an esterase from Emericella nidulans and Taralomyces emersonii (~1.6 kDa),
which is called microenzyme (Fan and Mattey, 1999). Further characterization of
the enzyme in fraction F would be very interesting for future application of
microenzymes.
Anti-oxidant activity
Anti-oxidant activity was evaluated by measuring reduction of DPPH.
Vitamin C (2 mM), vitamin E (2 mM), and BHT (2 mM) were employed as the
references whilst non-hydrolyzed snakehead fish collagen as the basal. The result
of radical scavenging challenge assay (Figure 5) suggested that the peptides
hydrolysate generated by fractions D demonstrated no or negligible anti-oxidant

Figure 5 Antioxidant Activity
activity, while peptides generated by fractions F showed up to 10% scavenging
activity. Interestingly, peptides hydrolysate generated by mixture of fraction D
and F showed up to 20% scavenging activity. This suggests that components of
the two enzyme fractions operate synergistically.
Anti-cholesterol activity
Anti-cholesterol potential of the peptides hydrolysate generated from the
snakehead skin collagen by the collagenases of B. licheniformis F11.4 was
evaluated by means of inhibition of the activity of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) (0.5-0.7 mgP/ml) using the well-known anticholesterol drug pravastatin as the reference. Samples employed were collagen
treated with enzymes of fraction D or F (in the presence and absence of 5 mM
PMSF or 5 mM EDTA) and non-hydrolyzed snakehead skin collagen (for the

13
basal activity). The result is presented in Figure 6. Interestingly, peptides
hydrolysate generated by fraction D displayed similar inhibition power of HMGR
activity to that of pravastatin. Further, treatment of fraction D with PMSF and/or
EDTA brought down the inhibition to the basal level. On the other hand, peptides
produced by fraction F demonstrated only 50% inhibition to HMG-CoA activity
in comparison to pravastatin. However, the activity of fraction F appeared to be
sustained upon treatment by both EDTA and PMSF. Fraction D is enigmatic
because inhibition of one or few of its enzyme components by EDTA and/or
PMSF resulted in impaired bioactive peptides production. Corresponding to the
characteristics of collagenases in fraction D, this result suggested that the
bioactive peptides were produced by the mixture of serine and/or metalloproteases, thus b1 and/or b2.
Fractions D and F were challenged to produce the snakehead fish skin
collagen peptides hydrolysate for inhibition of HMGR activities and radical
scavenging. HMGR is responsible for synthesis of mevalonate, and considered as
the key enzyme for biosynthesis of cholesterol and other non-steroidal isoprenoid
compounds (Arnaud et al., 2005). Therefore, controlling the HMGR activity may
lead to control the synthesis of cholesterol. Peptides hydrolysate produced by both
fractions D and F demonstrated full and 50% inhibition to HMGR activity,
respectively. However, fraction D was unable to produce the peptides hydrolysate
in the presence of EDTA and/or PMSF. On the other hands, fraction F still

Figure 6 Inhibition of HMG-CoA reductase
hydrolyzed the collagen to produce bioactive peptides hydrolysate in the presence
of the protease inhibitors. Combining the fractions D and F to produce bioactive
peptides hydrolysate with anti-cholesterol functionality could be an intelligent
option. This option may coincide with the finding of higher anti-oxidant activity
of peptides hydrolysate recovered from hydrolysis of collagen by the combined
fractions D and F. Thus, combined fractions D and F produced bioactive peptides
hydrolysate with high anti-cholesterol activity accompanied by anti-oxidant
activity, which leads to lower cholesterol level as well as prevention of radical
formation in the blood. This circumstance would be ideal to combat development
of cardiovascular diseases as functional food and drugs anti-CVD.

14

5 CONCLUSSION AND RECOMMENDATION
Conclusion
Secretion of collagenolytic enzyme from B. licheniformis F11.4 using acid
soluble collagen from snakehead fish skin as the inducer resulted in mixture of
collagenases with diverse characteristics. The enzymes were clustered into two
groups so called the fraction D and F. The two enzyme fractions are capable to
produce peptides hydrolysate that inhibits HMG-CoA activity and demonstrates
anti-oxidant activity. Combined activity of the two fractions produced peptides
hydrolysate to lower cholesterol and prevent radical formation, thus is potential
candidate for an anti cardiovascular diseases drug.
Recommendation
This study recommends continuing analysis of anti-oxidant using other
methods such as ferric reducing power anti-oxidant. Further the bioactivity study
can be pursued to analyze peptide as anti-cancer, and anti-hypertension.
Formulation of multi functional fish collagen as bioactive peptide hydrolysate
drink will give positive contribution in food functional sector.
Acknowledgements
The research was supported by Ministry of Research and Technology,
Republic of Indonesia, through funding incentive Sinas Research (Bioactive
peptides as anti-hypertension and anti-cholesterol) and Dexa Laboratories of
Biomolecular Sciences (DLBS). Thanks to Prof. F. Meinhardt from university of
Münster and Dr. Siswa Setyahadi from BPPT for kindly providing the B.
licheniformis F11.4.

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