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Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 Page ii
SCIENTIFIC BOARD
1. Prof. Dr. Abdul Wahab Jufri, University of Mataram
2. Dr. Elyzana Dewi Putrianti, Charite Universitaetmedizin, Berlin, Germany
3. Prof. Helmut Erdmann, University of Applied Sciences Flensburg, Germany
4. Dr. Imam Bachtiar, University of Mataram
5. Prof. James Gannon, University of Montana, USA (present address American
University of Sharjah, United Arab Emirates)
6. Dr. Lalu Rudyat Telly Savalas, University of Mataram
7. Assoc. Prof. Dr. Mian Muhammad Awais, Bahauddin Zakariya University,
Pakistan
8. Prof. Dr. Moh. Faried Ramadhan Hassanien, University of Zagazig, Egypt
9. Dr. Muhammad Roil Bilad, Nanyang Technological University, Singapore
(present address Universiti Teknologi Petronas Malaysia)
10. Dr. Saprizal Hadisaputra, University of Mataram
11. Dr. Syamsul Bahri, University of Mataram
12. Prof. Dr. Unang Supratman, University of Padjajaran
Technical Editors:
1. Baiq Nila Sari Ningsih, S.Pd.
2. Alfian Eka Utama
ISBN 9786021570425
Copyright: Penerbit FKIP Universitas Mataram
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 Page iii
PREFACE
Assalamu’alaikum warahmatullah wabarakatuh
It is my pleasure to be able to bring the International Conference on Mathematics
and Natural Sciences Proceeding to our readers. It took an extra effort, time and patience
to accomplish this proceeding and it involved reviewers from all over regions. I personally
thank to our reviewers and subsequently apologize for the delay in making this
proceeding available for you to read. It is largely due to the inevitably extensive reviewing
process and we persist on our initial idea to keep the proceeding both readable and
academically meet a higher standard.
This proceeding is presented in six sections: 1) Invited Speakers; 2) Physics; 3)
Mathematics; 4) Biology (including pharmacy and agriculture); 5) Chemistry; and 6)
General Education. All sections consist of papers from oral and poster presentation in
respective subject, including science and science education.
I hope that this proceeding may contribute in science and science education.
Wassalamu ‘alaikum warahmatullahi wabarakatuh
Lalu Rudyat Telly Savalas
Chief Editor
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 Page iv
TABLE OF CONTENTS
Page Scientific Board List ii Preface iii Table of Contents iv
Code Article Title SECTION I: INVITED SPEAKERS
Important Developments in Science Education: Next Generation Science Standards, Activity Theory, and Sociocultural Perspectives for Framing Science Teachingand Learning (Todd Campbell) .…………………………………… IS-1
The Educational Practices Framework: The Implementation of the ELPSA Model in West Nusa Tenggara (Tom Lowrie and Sitti Maesuri Patahuddin) ……………………………………………………………………………………………. IS-14
Real Work Is Better than Homework (Brian Coppola, Abstract only)………. IS-22
Antifungal Compounds Isolated from Endophytes against Japanese Oak wilt Pathogen, Raffaelea quercivora (Yoshihito Shiono) ………………………….. IS-23
Rapid Recovery of Degraded Reefs Following HighHuman Mortality from the Indian Ocean Tsunami (Andrew H. Baird, Abstract only)………………….. IS-27
House-farmed Edible-nest Swiftlets of Indonesia andMalaysia: Linked Studies of a New Domestication (Earl of Cranbrook’, Sarah Ball, W.L. Goh, Mohammad Saiful Mansor and Muhammad Rasul Abdullah Halim).. IS-28
Cryptic Species: Genetics and Systematics (Hoi-Sen Yong, Praphathip Eamsobhana, Phaik-Eem Lim, Sze-Looi Song and I. Wayan Suana)………….. IS-40
Fouling Control in Membrane Processes: Vibration and Surface Corrugation (Muhammad R. Bilad)……………………………………….…………………. IS-45
Theory of Metallic Nanoclusters (Elias Saion, Abstract only)………….…..….. IS-53 SECTION II: PHYSICS
OP-01 Fundamental Theories of Extra Dimensions and Neutrino (Muhammad Yusuf and Tasrief Surungan, Abstract only)……………………………………………… PHY-1
OP-02 Temporal Statistical Analysis of the Volcanic Eruption in Mt. Banda Api, Banda Islands, Maluku (Josephus Rony Kelibulin, Desi Kiswiranti, R.R Lokollo and H. Andayani)…………………………………………………………………………. PHY-2
OP-04 The Role of ZnPc Functional Layer in Detecting MMP3 Biomolecule Using QCM Sensor System (Djoko J. Santjojo and Masruroh, Abstract only)……... PHY-8
OP-05 Determination of in-situ Permeability by Using Stoneley Wave Characteristics (Kosim, Abstract only) …………………………………..………………… PHY-9
OP-06 Development of a Quake Catcher Device to Record Earth Quake Events (I Wayan Sudiarta and Made Sutha Yadnya, Abstract only)……...……………….. PHY-10
OP-07 Load Weight of Boat: Experimental Approach and Hydrostatic Calculations (Agus Dwi Catur, Sukartono, Sinarep and Masrun)…..…………. PHY-11
OP-08 Performance of Hinged Blade Savonius Turbine (Sinarep and Agus Dwi Catur) ……………………………………………………………………………………………………… PHY-18
OP-09 The Effect of Biogas Flow Rate Biogas in the Purification of Carbon Dioxide Process with Pumice Absorber (Arif Mulyanto, Nurchayati, Rudy Sutanto and Pandri Pandiatmi)……………………………….……………………………… PHY-26
OP-10 Characterization of Electric and Magnetic Properties ofBarium M-Hexaferrite Doped with Zinc (Aris Doyan, Susilawati and Ilham Halik)…….. PHY-30
OP-11 Synthesis by Coprecipitation Method and Characterization of Nickel-Doped Barium M-Hexaferrite (BaFe12O19)(Susilawati, Aris Doyan and Munib) ……………………………………………………………………………………………….…… PHY-37
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 Page ix
OB-03 The Use of Sawdust Storage Medium in Various Water Content to Maintain the Viability and Vigor of Shorea leprosula Mig and Dryobalanops oblongifolia Dyer Seed (Dyah Iriani, Siti Fatonah, Nita Anggraini and Kiswati) …….………………………………………………………………………
BIO-7 OB-04 Epidemiologi Study New Castle Disease Virus Blitar Isolate Based on
Fusion Protein (Sri Murwani, Dahliatul Qosimah, Rizki Arya Pradikta and Indah Amalia Amri) ……………………………………………………………………………….. BIO-15
OB-05 Pharmacokinetic Study of Rifampicin after Oral Intake of Noni (Morinda citrifolia Linn) in Male Rats (Farida Hayati, Ari Wibowo and Pradhani Daneswari, Abstract only) ……………………………………………………………………… BIO-19
OB-06 Growth Stand and Yield Performances of Some Modern Superior Maize Hybrid Varieties Grown under Different Population Densities on a Dryland (I Komang Damar Jaya, Sudirman and Jayaputra) ……………………. BIO-20
OB-07 Antibacterial Activities of Solanum torvum Fruit and Leaf Extract against Staphylococcus aureus and Pseudomonas aeruginosa (La Ode Muh. Julian, Khatamul Umam, Annisa Fitria and Hady Anshory, Abstract only).. BIO-25
OB-08 Bioanalytical Stability Test of Metformin Hydrochloride in Human Plasma Using Reversed-Phase HPLC-UV (Ari Wibowo, Nailatul Izzah and Vitarani D.A. Ningrum, Abstract only) …………………………………………………………………. BIO-26
OB-09 Antibacterial Activity of a Compound Isolated from Garcinia dulcis Leaves to Common Bacterial Pathogens (Hady Anshory Tamhid, Subagus Wahyuono and Triana Hertiani, Abstract only) ………………..……………………. BIO-27
OB-11 Anti Dandruff Shampoo from Kesum Leaves (Polygonum minus) Ethanol Extract (Dini Hadiarti, Abstract only) ………………………………………………………. BIO-28
OB-13 The Density of Meiobenthos from Mangrove Ecosystem, Inside the Buffer Zone of Giam Siak Kecil – Bukit Batu Biosphere Reserve, Bengkalis Regency, Riau Province (Radith Mahatma and Dewi Putri Arni) ……………… BIO-29
OB-14 The Effect of BAP and NAA on In Vitro Shoot Induction of Siam Orange (Citrus nobilis Lour.) from Kampar, Riau (Siti Fatonah, Mayta Novaliza Isda and Rahmahayu) ……………………………………………..……………………………… BIO-36
OB-15 Exploration of Indigenous Ammonia-Oxidizing Bacteria from Biological Pond on a Wastewater Treatment Plant of Urea Fertilizer Industry (Sri Wardhani, Moh. Rasyid Ridho, Arinafril, Susila Arita and Ngudiantoro)….. BIO-43
OB-16 Characterization and Development of Edible Film / Coating From Jackfruit Seed (Artocarpus heterophyllus Lam.) Starches for Preservation and Properties of Strawberry Fruits (W. Donald R. Pokatong and Elana Natania) …………………………………………………………………………………………………. BIO-50
OB-17 The Effect of Tristaniopsis obovata R.Br Leaf Extract on Spleen and Kidney Tissue Structure of Rattus norvegicusthat had Suffered from Urolithiasis (Wardatul Jannah, Tantia Shecilia, Yusfiati and Fitmawati, Abstract only) ……………………………………………….………………………………………… BIO-65
OB-18 Effect of Tristaniopsis obovata R.Br Leaf Extract on the Liver and Kidney Tissue Structure of Rat (Ratusnovergicus) (Yusfiati, Iriani Dyah and Fitmawati) ……………………………………………………………………………..………………. BIO-66
OB-19 Histopatological Assessment of Conjunctiva and Bronchus Mucous Membrane of Rattus norvegicusFollowing Citronella Oil Inhalation and Ultrasound WaveTreatment (Ardiana Ekawanti, Nurkaliwantoro and Warindi) ………………………………………….……………………………………………………… BIO-71
OB-20 Molasses as an Alternative Low-cost Medium for Production of Recombinant vp28-Antigen in Escherichia coli (Sukarne, Sulaiman N. Depamede, Muhamad Amin and Muhamad Ali) ….…………………………….…… BIO-78
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-78
OB-20 Molasses as an Alternative Low-cost Medium for
Production of Recombinant vp28-Antigen in Escherichia coli
Sukarne1, Sulaiman N. Depamede
1, Muhamad Amin
2, and Muhamad Ali
1*
1Laboratory of Microbiology and Biotechnology, Faculty of Animal Sciences, Mataram University,
Jl. Majapahit No. 62 Mataram, Indonesia
2Faculty of Fisheries, University of 45, Mataram, Indonesia
Abstract-Previous studies reported that VP28, which is a capsid protein of White Spot Syndrome Virus
(WSSV), showed to be a potential candidate for WSSV vaccine. However, the large-scale production of the
protein is hampered by the availability and expensive cost of culture media. Thus, this study was aimed at
finding alternative low-cost medium for growing bacterial hosts carrying plasmid for producing VP28
proteins fused with glutathione s-transferase (GST) enzyme which then indicated as GST.VP28 fusion
protein. The result showed that 7.5% molasses seemed to have better growth than commercial media (LB).
Other result also indicated that quality of GST.VP28 protein expressed by host in 7.5% molasses appeared to
be good. Ass indicated by its solubility. This result suggested that molasses medium can be an alternative
low-cost medium for large-scale production of GST.VP28 protein.
Keywords: white spot syndrome virus (WSSV), VP28, molasses.
1. Introduction Medium for culturing a bacterial host is important factors in order to produce targeted
extracellular metabolite products (Gopal, 2013). A common medium for growing a
bacterial host is Lysogeny Broth (LB) medium. This medium contains tryptone which are
peptide mixture from casein broken down by trypsin. However, the use of LB has several
weaknesses, including expensive cost, its availability, and low carbohydrate content which
cause limited growth (stationary phase is reached at OD600= 2 with bacterial biomass about
0.6 mg/mL medium). Therefore, studies to find alternative media with local ingredients is
urgently needed.
Several authors have reported the use of molases which is waste of sugar cane
industries as culture medium for bacteria due to its high nutrient contents including high
carbon sources (Bae and Shoda, 2004), nitrogen (Vohra, 2004), pantothenic acids, niacin,
vitamin B6 and cholin (Standbury and Whitaker, 1984). In addition, Keshk et al. (2006)
suggested that molasses had higher bacterial cellulose ratio compared to glucose medium.
With additional small amount of supplements, molasses can be an alternative for low-cost
media for growing common bacterial hosts such as E. coli.
E. coli BL21 has been widely used as a bacterial host to produce diverse recombinant
proteins (Du et al., 2006). This strain is generally preferred by many researchers due to
several advantages including fast growth, ability to grow at very high cell density with
very stable production of secondary metabolites (Shiloach and Fass, 2005; Tripathi 2009),
high expression capacity, very effective for translation of some rare codons, genetically
stable (Gopal, 2013), non-pathogen, and ability to accept new genes which encode target
proteins easily (Brock et al., 1994, Ali et al., 2005a,b,c
). In addition, compared to E. coli
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-79
K12, this strain produces less acetate during the culture periods which prevents the
decreasing of pH in growth media (Phue and Shiloach, 2004; Son et al., 2011).
Furthermore, multi-omics analysis showed that E. coli BL21 was more efficient in
producing proteins compared to E. coli K12 (Yoon et al., 2012).
This study was aimed at finding alternative low-cost medium for growing bacterial
hosts carrying plasmid for producing VP28 proteins, a capsid protein of White Spot
Syndrome Virus (WSSV) which has a promising potency as WSSV vaccine candidate. The
protein was fused with C-terminal of catalytic enzyme from Schistosoma juponicum
(glutathione S-transferase designated as GST) in pGEX gene fusion expression system.
The pGEX system is used widely for the overexpression of a wide variety of proteins in E.
coli, facilitating their soluble expression and purification using glutathione affinity
chromatography. Two common bacterial hosts (E. coli BL21 and E. coli BL21 plus RIPL
strains) were grown in different concentration of molasses and commercial (LB) media.
Then, growth of the bacterial hosts, protein quantity and quality expressed by both host
were compared to find out whether molasses could be alternative replacement of LB media
as well as the optimum concentrations.
2. Materials and Methods Preparation of LB medium
One-hundred millilitres LB agar consisted of 1 g tryptone powder, 0.5 mg yeast
extract, 1 g NaCl, and 100 distilled water (dH2O). Then, pH was adjusted to be 7.0 with 0.1
M Sodium hydroxide (NaOH) or hydrochloric acids (HCl). 1.5 g Agar was added to make
LB agar. Then the mixture was autoclaved and used for transformant inoculation or
bacterial expression.
Preparation of Molasses medium
Four different concentrations of molasses (3.5%, 5%, 7.5%, and 10%) were prepared
with additional of urea (8.5 mg/mL), KH2PO4 (3.1 mg/mL), and MgSO4 (1.71 mg/mL) and
distilled water (dH2O). pH of the medium was adjusted to 7.2 with NaOH or HCl, before
being sterilized and used for E. coli cultivation.
Analysis of gst.vp28 gene expression in E. coli BL21 and BL21 codon plus RIPL Strain
For gst.vp28 gene expression analysis, pGEX.VP28 plasmid was transformed by heat
shock method into competent cells of E. coli BL21 and BL21 codon plus RIPL strains
using standard method (Sambrook and Russell, 2001). Then, the transformant was plated
into LB-agar containing 50 µgmL-1
of ampicillin and incubated at 37oC for overnight. The
grown single colony was inoculated into 2 mL LB broth supplemented with 50 µgmL-1
of
ampicillin and incubated with shaking at 37oC for overnight. Then, 200 µl of the saturated
culture was inoculated into 2 mL LB broth supplemented with 50 µgmL-1
of ampicillin and
incubated at 37oC. Glycerol stock was also generated using the saturated culture and stored
at -80oC. Expression analysis (time of induction, IPTG concentration) were conducted to
explore the best condition for high level expression of GST.VP28. Cells were harvested by
centrifugation at 6,000 g at 4°C and the obtained pellet was diluted with 200 µl of PBS
buffer for SDS-PAGE analysis.
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-80
The use of molasses as a culture medium for GST.VP28 generation
Glycerol stock of E. coli BL21 and BL21 codon plus RIPL strains carrying
pGEX.VP28 plasmid were plated into LB-agar containing 50 µgmL-1
of ampicillin and
incubated at 37oC for overnight. The grown single colony was inoculated into 2 mL LB
broth supplemented with 50 µgmL-1
of ampicilin and incubated with shaking at 37oC for
overnight. Then, 200 µl of the saturated culture was inoculated into 2 mL LB broth or
molasses media supplemented with 50 µgmL-1
of ampicilin and incubated at 37oC until
OD600 = 0.5. After that, IPTG was added into the culture and continued for cultivation. The
level of IPTG and incubation time were obtained from the best GST.VP28 expression
analysis. Cells were harvested by centrifugation at 6,000 g at 4°C and the obtained pellet
was diluted with 200 µl of PBS buffer for SDS-PAGE analysis.
Analysis of fusion protein solubility
To measure the amount of soluble and un-soluble fraction, the pellet obtained is
suspended in 160 mL of lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and
1.8 mM KH2PO4, 1 mM PMSF, pH 7.3) and lysed by sonication (10 x 30 s pulse with 45 s
interval). The lysate obtained is centrifuged at 12, 000 g for 15 min. After centrifugation,
the resulting supernatant is transferred into a fresh tube and subsequently compared the
fusion protein contents in the supernatant with the pellet using SDS-PAGE.
3. Results and Discussion Potency of molasses as LB media replacement
Due to its nutritional contents, molasses which is by product of refining sugarcane is
potential sources of culture media for microorganism (Shasaltaneh et al., 2013). The
molasses contains several mixed sugars, including glucose, sucrose, and fructose.
Akaraonye et al. (2012) stated that the molasses is considered to be a promising feedstock
for biorefinery due to its rich sugar content and cost-effectiveness. Culturing two E. coli
bacterial hosts carrying pGEX.VP28 recombinant plasmid with molasses media showed
that total biomass productions of E. coli BL21 and E. coli BL21 codon plus in several
concentration of molasses was presented in Fig. 1.
Figure1. Growth of E. coli BL21 and E. coli BL21 codon plus (C+) grown in molasses. 1 = E. coli
BL21 in 3,5 % molasses; 2 = E. coli BL21 C+ in 3,5% molasses; 3 = E. coli BL21 in 5% molasses; 4 = E.
coli BL21 C+
in 5% molasses, 5= E. coli BL21 in 7.5% molasses; 6 = E. coli BL21 C+ in 7.5% molasses; 7
= E. coli BL21 in 10% molasses; 8 = E. coli BL21 C+ in 10% molasses; 9 and 10 = E. coli BL21 and E.
coli BL21 C+
in LB.
The figure indicated that the weight of wet biomass of both bacteria was increased
consistently from low concentration of molasses (3.5%) to 7.5%. However, the use of 10%
0.00002.00004.00006.00008.0000
10.000012.000014.000016.000018.000020.0000
1 2 3 4 5 6 7 8 9 10
Biomass (mg)
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-81
of molasses reduced these hosts biomasss, indicating that the molasses concentration is
harmful for E. coli gowth. This result might be caused by growth inhibitory effect of sugar
component of the molasses, a decrease in hydrolysis rate of sugar, or osmotic effect of
sugar high concentration. Batch cultures of E. coli in the presence of excess glucose or
glycerol produce acidic fermentation by-products, in particular acetate (Eiteman and
Altman, 2006; Martinez-Gomez, 2012). Acetate is a known inhibitor of biomass and
recombinant protein production (Kleman and Strohl, 1994; Mey et al., 2007).
The most interesting result was higher biomass production of both bacterial hosts (E.
coli BL21 and BL21 codon plus) cultured in molasses medium compared in LB medium.
This might be prior to the availability of diverse sugars including glucose, sucrose, and
fructose are more in the molasses medium as carbon sources (Akaraonye et al., 2012). As
stated previously, LB has low carbohydrate content which cause limited growth in bacteria.
It is commonly known that the stationary phase of bacteria in the LB medium is reached at
OD600 = 2 with bacterial biomass about 0.6 mg/mL medium. The addition of nitrogen
sources such as urea and other nutrients such as Mg2SO4 and KH2PO4 will also increase the
utilization of molasses medium.
To study the effect of molasses media on expression level of recombinant protein, the
targeted GST.VP28 protein expressed in both hosts was measured roughly using SDS-
PAGE, and the result was presented in Fig. 2. The GST.VP28 protein synthesized is
reflected by the appearance of the one major product with a 45.9 kDa molecular weight
identical to that of the GST (26 kDa) (Anonim, 2012; Fei et al. 2015) and VP28 ( 19.9
kDa). Seok et al. (2004) reported that there are some variability of VP28 gene size which
then caused the variability in molecular weight of VP28 protein. Furthermore, Tang et al.
(2007) reported that the molecular weight of VP28 is 22 kDa and VP28 from Brazilian
WSSV has 21 kDa molecular weight (Braunig et al. 2011).
SDS-PAGE results in Fig 2 revealed that the intensity of the 45.9-kDa band
corresponding to the GST.VP28 synthesized using 5% and 7.5% molasses was higher than
that using other level of molasses. These findings are consistent with generated biomass
weight (Fig. 1), in which the use of 5% and 7.5% molasses produced higher total biomass
of E. coli BL21 and BL21 codon plus then using other concentration.
Figure 2. GST.VP28 produced in several molasses concentration. M = protein marker, 1 = E. coli BL21
in molasses 3,5% (- IPTG), 2 = E. coli BL21 in molasses 3.5%, 3 = E. coli BL21 C+ in molasses 3.5%, 4 = E.
coli BL21 in molasses 5%, 5 = E. coli BL21 C+ in 5% molasses, 6 = E. coli BL21 in molasses 7,5%, 7 = E.
coli BL21 C+
in molasses 7.5%, 8 = E. coli BL21 in molasses 10%, and 9 = E. coli BL21 C+ in molasses
10%.
45,9 kDa
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-82
Optimal biomass growth and protein expression from both bacterial hosts in 5% and
7.5% molasses might indicate that nutrition content in molasses was very suitable for
growing the bacterial hosts. Molasses concentration lower that 5% might provide un-
sufficient amount of nutrient, meanwhile higher concentration seemed to effect medium
viscosity which later influence dissolved oxygen in the medium. In addition, higher
molasses concentration also might pH of the medium during culture periods (Agarwal,
2005).
Solubility of GST.VP28 recombinant protein
Thereafter, the quality of the expressed protein was assessed by checking protein
solubility. Figure 3 represented the expressed GST.VP28 protein from E. coli BL21
cultured in 5% and 7% molasses medium based on SDS-PAGE.
Figure 3. Soluble and unsoluble fraction of GST.VP28 generated by E. coli BL21 in molasses 5% dan
7.5%. M = marker, 1 = molases 5% (- IPTG), 2 = total protein in molases 5%, 3= soluble fraction in molases
5%, 4 = un-soluble fraction in molasses 5%, 5 = molasses 7.5% (- IPTG), 6 = total protein in 7.5% molasses,
7 = soluble fraction in molasses 7.5%, 8 = un-soluble fraction in molasses 5%.
Figure 3 showed that the amount of protein was higher in 5% molasses medium
compared to 7.5% molasses medium. However, a tiny amount of the expressed protein was
available in the supernatant after sonication. On the other hand, almost all of the expressed
protein generated using 5% molasses was available as insoluble form which known as
inclusion body. Therefore, based on its solubility, E. coli BL21 grown in 7.5% molasses
medium produced GST.VP28 with better solubility than generated with 5% molasses.
4. Conclusion E. coli BL21 cultured in molasses medium could produce the same amount of
recombinant protein GST.VP28 as LB medium does. This result suggested that molasses
medium could be an alternative medium to produce the recombinant protein in large scale
with less cost.
Acknowledgment
The authors were grateful to Prof Hideo NAKANO, Assist. Prof. Takaaki KOJIMA,
and Assist. Prof. Jasmina DAMNJANOVIC (Laboratory of Molecular Biotechnology
Nagoya University) for useful advice and helpful discussion. This work was supported by
grants from Directorate General of Higher Education Indonesian Ministry of Research,
Technology and Higher Education, Republic of Indonesia.
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-83
References
Agarwal, L., Isar, J., Meghwanshi, G.K., and Saxena, R.K. 2005. A Cost Effective Fermentative
Production of Succinic Acid From Cane Molasses and Corn Steep Liquor by Escherichia coli.
Journal of Applied Microbiology.
Akaraonye E, Moreno C, Knowles JC, Keshavarz T, Roy I. 2012. Poly(3‑hydroxybutyrate)
production by Bacillus cereus SPV using sugarcane molasses as the main carbon source.
Biotechnol J 7:293– 303.
Ali, M., Suzuki H., Fukuba T., Jiang X., Nakano H., and Yamane T. 2005a. Improvements in the
Cell- free production of functional antibodies using cell extract from protease-deficient
Escherichia coli mutant. J. Biosci., Bieng., 99, 181-186.
Ali, M., Hasan R., and Nakano H. 2005b. Periplasmic endoprotease, DegP, as a potential obstacle
to recombinant protein production in Escherichia coli cell-free technology. Proceedings of
14th Indonesian Sci. Conference, 121-128.
Ali, M., Hasan R., and Nakano H. 2005c. Escherichia coli mutants construction using combination
of one step chromosomal disruption-P1 phage transduction methods. Proceedings of 14th
Indonesian Sci. Conference, 89-96.
Ali, M., Hitomi K., and Nakano H. 2006. Generation of monoclonal antibodies using simplified
single cell reverse transcription-polymerase chain reaction and cell-free protein synthesis.
J. Biosci., Bieng., 101, 284-286.
Anonim, 2012. GST Tag ELISA Detection Kit: Technical Manual No. TM0521. GenScript. Cat.
No. L00411
Bae, S., and Shoda, M. 2004. Bacterial Cellulose Production by Fed-Batch Fermentation in
Molasses Medium. Journal of Biotechnology Progress. Vol. 20, P. 1366 – 1371.
Bertani, G. 1952. "Studies on Lysogenesis. I. The mode of phage liberation by lysogenic
Escherichia coli." J. Bacteriology, 62:293-300
Bertani, G. 2004. "Lysogeny at mid-twentieth century: P1, P2, and other experimental systems." J
Bacteriology, (186):595-600
Braunig, P., Rafael, D.R., Caroline, H.S., Mariana, B., Patricia, H.S., Edmundo, C. Grisard and
Aguinaldo R. Pinto. 2011. Molecular cloning and recombinant expression of the VP28
carboxyl- terminal hydrophilic region from abrazilian white spot syndrome virus isolate.
Brazilian archives of biology and technology vol.54, n. 2: pp. 399-404.
Brock, T.D., M.T. Madigan, J.M. Martinko & J. Parker. 1994. Biology of microorganism. 7th ed.
Prentice-Hall, Inc., New Jersey: xvii + 909 hlm.
Du, H., Xu, Z., Wu, X., Li, W., and Dai, W. 2006. Increased resistance to white spot syndrome
virus in Procambarus clarkii by injection of envelope protein VP28 expressed using
recombinant baculovirus. Aquaculture. Vol. 260, P. 39 – 43
Eiteman MA, Altman E. 2006. Overcoming acetate in Escherichia coli recombinant protein
fermentations. Trends Biotechnol 24:530–536 8.
Fei, Dongliang., Zhang, H., Diao, Q., Jiang, L., Wang, Q., Zhong, Yi., Fan, Zhaobin
and Ma., M. 2015. Escherichia coli, and Immunogenicity of Recombinant
Chinese Sacbrood Virus (CSBV) Structural Proteins VP1, VP2, and VP3. Plos
One Research Article.
Gopal, J.G., and Kumar, A. 2013. Strategies for the Production of Recombinan Proteinin
Escherichia coli. Protein Journal. (32) p, 419 – 425.
Gustafsson, C., Govindarajan, S., Minshull, J. 2004. Codon Bias and Heterologous Protein
Expression. Trends Biotechnol, 22:346–353.
Keshk, S., Razek T., and Sameshima, K. 2006. Bacterial Cellulose Production from Beet Molasses.
African Journal of Biotechnology. Vol. 5, No7
Kleman GL, Strohl WR (1994) Acetate metabolism by Escherichia coli in high-cell-density
fermentation. Appl Environ Microbiol 60:3952–3958 10.
Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015
ISBN 9786021570425 BIO-84
Kopanic, J.L., Al-Mugotir, M., Zach, S., Das, S., Grosely, R. and Sorgen, P.L. 2013. An
Escherichia coli Strain for Expression of the Connexin45 Carboxyl Terminus
Attached to the 4th Transmembrane Domain. Frontiers in Pharmacology.
Martínez-Gómez K, Flores N, Castañeda HM, Martínez-Batallar G, Hernán-dez-Chávez G,
Ramírez OT et al (2012) New insights into Escherichia coli metabolism: carbon scavenging,
acetate metabolism and carbon recycling responses during growth on glycerol. Microb
Cell Factories 11:46
McNulty DE, Claffee BA, Huddleston MJ, Porter ML, Cavnar KM, Kane JF: Mistranslational
Errors Associated with the Rare Arginine Codon CGG in Escherichia coli . Protein Expr
Purif 2003, 27:365–374
Mey MD, Maeseneire SD, Soetaert W, Vandamme E (2007) Minimizing acetate formation in E.
coli fermentations. J Ind Microbiol Biotechnol 34:689–700
Phue J-N, Shiloach J. 2004. Transcription levels of key metabolic genes are the cause for di-erent
glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109). J Biotechnol 109:21–
30. [Recombinant Proteins and Host Cell Physiology]
Rosano, G.L., and Ceccarelli, E.A. 2009. Rare Codon Content Affects the Solubility of
Recombinant Proteins in a Codon Bias-adjusted Escherichia coli Strain. Microbial
Cell Factories 8:41
Shasaltaneh MD., Moosavi-Nejad Z., Gharavi S., and Fooladi J. 2013. Cane molasses as a source
of precursors in the bioproduction of tryptophan by Bacillus subtilis. Iranian J. Microbiology,
5, 285- 292.
Sahdev, S., Khattar, S.K., and Saini K., 2008. Production of active eukaryotic protein through
bacterial expression systems: a review of the existing biotechnologies strategies. Mol. Cell.
Biochem., 307, 249-264.
Sambrook, J. & D.W. Russell. 2001. Molecular Cloning: A Laboratory Manual. 3rd
ed. CSHL
Press, New York: xxvii + 18.
Seok, S.H., Park, J.H., Cho, S.A., Baek, M.W., Lee, H.Y., Kim, D.J., Park, J.H., 2004. Cloning and
sequencing of envelope proteins (VP19, VP28) and nucleocapsid proteins (VP15, VP35) of a
white spot syndrome virus isolate from Korean shrimp. Dis Aquat Organ. 60, 85-88.
Sezonov, G., Petit, J.D., and D‘Ari R. 2007. Escherichia coli Physiology in Luria-Bertani Broth.
Journal of Bacteriology, P. 8746 - 8749
Shiloach J, Fass R. 2005. Growing E. coli to high cell density—A historical perspective on method
development. Biotechnol Adv., 23:345–357.
Tang, X.; Wu, J.; Sivaraman, J.; Hew, C.L. 2007. Crystal structures of major envelope proteins
VP26 and VP28 from white spot syndrome virus shed light on their evolutionary
relationship. J. Virol., 81, 6709-6717.
Tripathi NK. 2009. High yield production of heterologous proteins with Escherichia coli. Def. Sci.
J., 59:137–146.
Son Y-J, Phue J-N, Trinh LB, Lee SJ, Shiloach J. 2011. The role of Cra in regulating acetate
excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth. Microb
Cell Factories 10:52.
Sorensen, H.P. and Mortensen, K.K. 2005. Advanced genetic strategis for recombinant expression
in Escherichia coli. J Biotechnol, 115,113-128.
Standbury PF., and Whitaker A. 1984. Principles of Fermentation Technology. Pergamon Press.
Villaverde, A., and Cario, M.M. 2003. Protein aggregation in recombinant bacteria: biological role
of inclusion bodies. Biotechnol. Left, 25, 1385-1395.
Vohra, A. And Satyanarayana, T. 2004. A Cost-effective Cane Molasses Medium for Enhanced
Cell- bound Phytase Production by Pichia anomala. Journal of Applied Microbiology,
97, 471–476.
Yoon SH, Han M-J, Jeong H, Lee CH, Xia X-X, Lee D-H et al (2012) Com-parative multi-omics
systems analysis of Escherichia coli strains B and K-12. Genome Biol 13:R37.