lombok island, indonesia, november 4-5, 2015

Proceedings International Conference on Mathematics, Sciences and Education, University of Mataram 2015 Lombok Island, Indonesia, November 4-5, 2015

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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


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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


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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


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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

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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

[email protected]

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


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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.


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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)


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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


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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.


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