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1	Biokimia Komputasi	ABS-4
COMPUTATIONAL STUDY OF DIRECTED POINT MUTATION ON THE CATALYTIC RESIDUE OF HALOACID DEHALOGENASE FROM Pseudomonas aeruginosa ITB1 Nurul Nanda Khoiriyah, Enny Ratnaningsih, Ihsanawati Department of Chemistry, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jalan Ganesa 10, Bandung 40132, Indonesia Abstract Organohalide used as pesticides are one of the dangerous pollutants because they are difficult to be decomposed and toxic to organism. Dehalogenase-producing bacteria are one of potential solution to restore organohalide contaminated areas because of its ability to catalyses the decomposition of organohalides into other non-toxic compounds. Pseudomonas aeruginosa ITB1 is a local bacterium that produces dehalogenase (named as Paed-d). This enzyme catalyses the degradation of monochloroacetic acid (MCA) to yield environmentally friendly hydroxy-ethanoic acid. In order to determine the tertiary structure of the Paed-d molecule and its interaction with MCA, this research was carried out using a computational method of directed point mutation on the catalytic site of Paed-d. The Paed-d tertiary structure obtained from the Swiss Model web service is the best structure selected for further analysis. Computational and literature studies on two groups of haloacid dehalogenase enzymes, namely group I and group II, indicated that Paed-d turns out to be a group II haloacid dehalogenase. Paed-d follows a homo-dimer structure with each monomer has a Rossmann-fold shaped core domain consisting of 6 &#946--sheet strands flanked by 5 &#945--helices with a cap domain consisting of 4 &#945--helical bundle. The Paed-d active site resides at the junction of the core domain and cap domain, with the D7 nucleophilic residue located at the end of the first &#946--sheet of the N-terminal end. Further analysis suggested that the Paed-d active site residues consist of D7, V8, F9, T11, R44, S121, N122, G123, K152, H178, and D181. The D7A mutation caused hydrogen bonds loss between MCA and D7, N122, and G123, and caused the Paed-d-MCA complex structure to become less stable, less compact, and more exposed to solvents. This could possibly cause Paed-d D7A to lose part or all of its activity as indicated by graph data of molecular dynamic simulation potential energy, radius of gyration, and SASA (Solvent-Accessible Surface Area). On the other hand, S121P mutation caused MCA to lose two hydrogen bonds with S121 and the Paed-d secondary structure changed from &#946--sheet to coil at residues 119-121 and 141-144. However, this S121P mutation did not cause a significant change in Paed-d-MCA complex structure. From the beginning, D14 did not act as a catalytic residue and was not proven to replace D7 as a nucleophile in Paed-d D7A because it was not resided in the catalytic pocket and was located far from the position of the MCA ligand. Finally, the catalytic mechanism was proposed based on the literature study and the results of this study. The result of computational studies is a potential first step to study the relationship between the structure and function of the haloacid dehalogenase. Keywords: haloacid dehalogenase, molecular dynamics simulation, monochloroacetic acid, in silico mutation, Pseudomonas aeruginosa Share Link | Plain Format | Corresponding Author (Nurul Nanda Khoiriyah)

2	Biokimia Komputasi	ABS-5
Role of Tryptophan W201 and W202 Residues of BaqA\(\bf \triangle\)C \(\bf \alpha\)-Amylase on Substrate Binding Diandra Sekar Annisa, Ayra Ulpiyana, Fernita Puspasari, Reza Aditama, Dessy Natalia Biochemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology. Abstract BaqA is an \(\alpha\)-Amylase from \(\it Bacillus\) \(\it aquimaris\) MKSC 6.2 that is isolated from a soft coral \(\it Sinularia\) \(\it sp\). which has the ability to degrade several raw starches, such as corn, rice, sago, cassava, and potato. Interestingly, BaqA has no starch binding domain (SBD) which is commonly found in raw starch degrading enzymes. Instead, \(\it in\) \(\it silico\) analysis suggested the presence of a surface binding site (SBS) consisting of double tryptophan (Trp201 and Trp202) in domain A and sugar-tongs residue (Tyr400) in domain C. The aim of this research was to study the potential role of tryptophan as SBS by employing molecular docking and molecular dynamics approaches. In this study, BaqA\(\triangle\)C which is a truncated BaqA lacking the C terminal 34 residues was used which has more solubility in water. Analysis was carried out to study the effect of the substitution of Trp201 and Trp202 into alanine (W201A, W202A, W201A/W202A) and phenylalanine (W201F) on affinity with substrates and protein stability. The BaqA\(\triangle\)C modeling was successfully carried out through the I-TASSER server with close to good results. It was found that mutations on BaqA\(\triangle\)C did not significantly affect protein stability. However, it leads to a decrease in the movement of residues at loop III located in domain B. Therefore, these results suggested that residues W201 and W202 might help to maintain the flexibility of loop III in domain B which is near the active center region of the protein. In addition, these studies reveal that the analog substrate could bind to the protein surface through interaction with the aromatic residue W201. Keywords: BaqA\(\triangle\)C, surface binding site (SBS), double tryptophan, in silico mutation, molecular docking, molecular dynamics Share Link | Plain Format | Corresponding Author (Diandra Sekar Annisa Diandra Sekar Annisa)

3	Biokimia Komputasi	ABS-15
Design of a multiepitope vaccine from the HPV E1 and E2 proteins with an immunoinformatics and molecular dynamics approach Rizarullah (a), Reza Aditama (a), Ernawati Arifin Giri-Rachman (b), Rukman Hertadi (a*) a) Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jl. Ganesa No. 10, Bandung 40132, Indonesia b) Genetics and Molecular Biotechnology Research Division, School of Life Sciences and Technology, Bandung Institute of Technology, Jl. Ganesa No. 10, Bandung 40132, Indonesia Abstract Cervical cancer caused by human papillomavirus (HPV) is the fourth leading cause of death among women. Several commercial prophylactic vaccines are used to prevent HPV infection. However, these prophylactic vaccines have no therapeutic effect against previous infections of HPV as viral strategies to evade the immune system can increase HPV infection. Thus, this study aimed to develop a multiepitope vaccine based on the target proteins E1 and E2 of HPV 16, 18, 45, and 52 using bioinformatics. In this study, cytotoxic T lymphocyte (CTL) and helper T Lymphocyte (HTL) epitopes were predicted and their allergenicity, toxicity, and antigenicity were evaluated. The epitopes were constructed into a multiepitope vaccine using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled and refined with alphaFold 2.3 and GalaxyRefine, respectively. The quality of the tertiary structure model was evaluated by Ramachandran plots and Prosa-web and revealed that the vaccine construct had a good structural conformation. Molecular docking analysis and dynamic molecular simulation were performed to validate the interaction of the vaccine-TLR4 complex and the stability of the formed interaction, showing that the multiepitope vaccine interacted well with Toll Like Receptor 4 (TLR4). Vaccine-TLR4 binding affinity was calculated using MMGBSA, resulting in a binding value of -143.6992 kcal/mol. These results suggest that the proposed vaccine can be considered effective as a therapeutic vaccine. However, further in vitro and in vivo testing is needed to validate the effectiveness of the vaccine. Keywords: Cervical cancer- Human papillomavirus- Immunoinformatics- Multiepitope Vaccine- Molecular docking- Molecular dynamic Share Link | Plain Format | Corresponding Author (Rizarullah Rizarullah)

4	Biokimia Komputasi	ABS-43
Design Peptide to Disrupt Proper Orientation of NS1 DENV Dimerization Studied by Molecular Dynamics Simulation Asep Iin Nur Indra, Ihsanawati, Rukman Hertadi, Reza Aditama Institute Technology of Bandung Abstract The development of peptides for the treatment of dengue hemorrhagic fever has been widely reported to be more effective and less toxic compared to organic compound drugs. The target protein used as therapy is mostly non-structural proteins (NS) DENV because they have important role in the life-cycle of the virus. The NS1 protein, which is a multifunctional protein, has become the newest target for viral therapy recently due to its involvement in viral replication and pathogenesis. The active form of this protein is in dimeric and hexameric conformation. In this study, the inactivation of the NS1 protein will be carried out by interfering with the dimerization process of this protein. In order to prevent or change the orientation of NS1 dimerization, a peptide with a certain sequence will be designed. Interfacial interactions on the NS1 DENV protein dimer occur in the &#946--roll region (sequence 1-29) through ionic interactions in addition to hydrogen bonding and hydrophobic interactions. Docking results showed that the designed peptide with the sequence of nineteen amino acid was able to interact closely with the beta-roll region of the monomeric NS1 protein. The molecular dynamics simulation (MD) study showed that this binding can disorient the dimeric formation of NS1 protein, thereby preventing the formation of the functional form of dimeric NS1 proteins Keywords: NS1 protein, DENV, peptide, Molecular dynamics simulations Share Link | Plain Format | Corresponding Author (Asep Iin Nur Indra)

5	Biokimia Komputasi	ABS-53
Biochemical Properties Characterization of Recombinant alpha-amylase BmaN1 from Bacillus megaterium NL3 Muhammad Akbar Thufail, Fina Khaerunnisa Frima, Ihsanawati, Reza Aditama, Dessy Natalia* (a) Chemistry, Bandung Institute Technology Jalan Ganesha 10, Bandung 40132, Indonesia *dessynatalia[at]itb.ac.id Abstract Keywords: alpha-Amylase- BmaN1- Characterization- Biochemistry Share Link | Plain Format | Corresponding Author (Muhammad Akbar Thufail)

6	Biokimia Lingkungan	ABS-9
Bioconvertion of PKO to Rhamnolipid by Mutant Pseudomonas stutzeri BK-AB12 MT-03 and Its Aplication as Environmentally Friendly Corrosion Inhibitor Agents Rikzan Zuhdi, Rukman Hertadi Institut Teknologi Bandung Abstract Steel is primary material used for infrastructure because it is hard and highly resistant. However, steel can corrode when it comes into contact with the environment. Giving synthetic corrosion inhibitors as the main solution to prevent corrosion can be toxic, making things worse. For this reason, this study aims to examine the inhibitory potential of rhamnolipid bioconverted from Palm Kernel Oil (PKO) by mutant Pseudomonas stutzeri BK-AB12 MT-03 on steel material. In the early stages, the composition of PKO as the main substrate was tested using GC-MS. Then, the production and extraction of rhamnolipids were carried out using Pseudomonas stutzeri BK-AB12 MT-03. Then, characterization of the FTIR functional groups, physicochemical tests, and rhamnolipid stability were carried out by determining the decrease in surface tension of water and testing the potential for corrosion inhibitors using the weight loss method. The results show that the main constituents of PKO are natural lauric acid, myristic acid, and 9,10-dihydroxy decanoic acid. PKO was successfully converted into rhamnolipid as a brownish paste with a gain of 464.0 +- 37.8 mg/L of the medium. Analysis using FTIR showed the presence of aliphatic O-H, C-H vibrations, unsaturated C-H, and C=O groups which is typical for rhamnolipid vibrations. The CMC value of the rhamnolipid sample was found to be 52.8 mg/L with a decrease in surface tension of up to 27.80 mN/m. The surface activity of rhamnolipids is known to be stable against increasing salt concentration, temperature, and pH, indicating the potency of rhamnolipids under various environmental conditions. The percent inhibition efficiency (%EI) of rhamnolipids showed a maximum value of 59.75% at a concentration of 300 ppm. The attachment of rhamnolipids to the steel surface follows the Langmuir isotherm which indicates monolayer adhesion. The results showed that rhamnolipid can be used as a corrosion inhibitor for steel materials. Keywords: corrosion inhibitor, eco-friendly, PKO, Pseudomonas stutzeri BK-AB12 MT-03, rhamnolipids. Share Link | Plain Format | Corresponding Author (Rikzan Zuhdi)

7	Biokimia Lingkungan	ABS-16
Graphene quantum dots functionalised with rhamnolipid produced from bioconversion of palm kernel oil by Pseudomonas stutzeri BK-AB12MT as a photocatalyst** Samuel Ivan, Fera Faridatul Habibah, Atthar Luqman Ivansyah, Rukman Hertadi Bandung Institite of Technology Abstract Methylene blue (MB) is a common organic dye found in textile wastewater and can harm the environment. Rhamnolipid-functionalized graphene quantum dots (RL-GQDs) are a newly developed eco-friendly photocatalyst to degrade MB. This photocatalyst is synthesized from graphene quantum dots (GQDs) and rhamnolipid. GQDs are already promising visible-light photocatalysts to degrade organic dyes. However, GQDs are not promising photocatalysts due to their reusability and photocatalytic performance. In this work, we used rhamnolipid to modify GQDs^ structure and enhance their photocatalytic performance. The rhamnolipid used in this work was produced from bioconversion of palm kernel oil by mutated bacterial cells of Pseudomonas stutzeri BK-AB12MT. Meanwhile, GQDs were synthesized using the bottom-up method by pyrolysing citric acid. Transmission electron microscopy and Fourier-Transform Infrared spectroscopy were used to characterize these hybrid materials. These characterization techniques verified the formation of RL-GQDs. To prove the photocatalytic performance of RL-GQDs, we investigated the photocatalytic activity under visible light compared to some common photocatalysts, such as zinc oxide and titanium dioxide. Our findings showed that RL-GQDs could be applied as an eco-friendly photocatalyst to replace TiO2 with a degradation efficiency of 59 (percent) aproximaly 3 (percent) under visible light irradiation, higher than TiO2. Keywords: Methyleb Blue Degradation, Photocqtalyst, Rhamnolipid Functionalized Quantum Dots Share Link | Plain Format | Corresponding Author (Fera Faridatul Habibah)

8	Biokimia Lingkungan	ABS-37
cDNA Amplified from RNA of Diatom Cyclotella striata TBI Cultivated Under Various Light Conditions Nurfarida Ulfah, Sari Dewi Kurniasih Indrawan, Alfredo Kono, Yanti Rachmayanti, Zeily Nurachman* Biochemistry Division, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jalan Ganesa 10, Bandung 40132, Indonesia *zeily[at]itb.ac.id Abstract Lipids play a crucial role in microalgae and their metabolism was regulated by several enzymes related to their synthesis and degradation. Cyclotella striata TBI is one of tropical diatom that uses lipid as food storage component. Lipid accumulation increases when exposed to external stress factors such as high light intensity and temperature, and it could be identified from the expression at the level of RNA transcript, but RNA is an unstable compound, so cDNA synthesis is carried out as an alternative. Until now, synthesis of cDNA from diatom C. striata TBI RNA grown indoor and outdoor has not been carried out. The aim of this study was to identify cDNA by RT-PCR method from diatom C. striata TBI RNA cultivated under various light conditions. Methods of this study included cultivating C. striata TBI indoor and outdoor in the modified seawater medium, total lipid extraction, total RNA isolation, and cDNA synthesis from total RNA. The results showed that the initial C. striata TBI cell density of 4.810^{5} cells mL^{-1} increased to 2.25\times10^{6} and 1.05\times10^{6} cells mL^{-1} both indoor and outdoor cultures with cultivation for 8-9 days. This is equivalent to specific growth rates of 1.78 and 1.66 d^{-1}, or cell productivity of 2.69 and 1.52\times 10^{6} cells mL^{-1} d^{-1}, or biomass productivity of 71.27 and 47.95 mg L^{-1} d^{-1}. The total lipid content of \it C. striata TBI obtained from indoor and outdoor cultures was 32 and 49% (w/w) respectively, which equates to lipid productivity of 2.2 and 2.3 mg L^{-1} d^{-1}. RNA from \it C. striata TBI cultures both indoor and outdoor in logarithmic and stationary phases was identified by the presence of bands measuring 1400 bp, 900 bp, 250 bp which indicated 28S, 18S, and 5S RNA respectively, with concentrations ranging from 0.8 to 1.0 mg mL^{-1}. cDNA synthesis was successfully identified from the presence of internal controls bands ACTB and rbcL which were 500 bp and 750 bp, respectively. cDNA from these conditions has a concentration range of 1.0-1.6 mg mL^{-1}. cDNA from \it C. striata TBI RNA both from indoor and outdoor cultures has high purity and concentration. Keywords: C. striata TBI, lipid, indoor, outdoor, RNA, cDNA Share Link | Plain Format | Corresponding Author (Nurfarida Ulfah)

9	Biokimia Lingkungan	ABS-42
Application of ectoine as a co-solvent in esterification reactions in biodiesel synthesis Alfina Marwiyah, Rukman Hertadi Chemistry, Faculty of Matemathics and Natural Sciences, Bandung Institute of Technology Abstract Biodiesel is an alternative fuel produced from renewable natural materials such as vegetable and animal oils, specially formulated for diesel engine fuel, with the advantages of high lubricating power, being environmentally friendly, safe, and non toxic. Biodiesel synthesis involves an esterification process with the help of a lipase enzyme catalyst. Lipase (triacylglycerol hydrolase) catalyzes the reaction of ester group formation to produce monoalkyl esters of fatty acids. The synthesis process requires a lipase that has stability against extreme conditions and organic solvents, especially alcohols. Ectoine is one of the osmolytes found in several types of halophilic bacteria that can protect proteins from heating, freezing, and chemical contact. This research will study the esterification activity with the addition of ectoine as a co solvent in the esterification reaction in biodiesel synthesis. Ectoine production from the halophilic bacteria Halomonas elongata BK AG25 was optimized by the osmotic shock technique grown on MM63 media. The bacteria were then inoculated on the same media with a high salt concentration (osmotic upshock) to produce ectoine and then transferred into distilled water with a low salt concentration (osmotic downshock) to release the ectoine produced. The ectoine obtained was then analyzed for its structure using NMR and HPLC. The success of ectoine as a lipase coprocessor was tested using the titration method to determine the esterification activity between oleic acid and ethanol in n hexane solvent by varying the concentration of ectoine used. Ectoine was successfully produced and was able to increase the esterification activity in the ratio of 2:1 to ethanol. Ectoine is also known to be able to maintain lipase stability at temperatures up to 50oC and at a more alkaline pH. Keywords: Ectoine, lipase, esterification, activity Share Link | Plain Format | Corresponding Author (Alfina Marwiyah)

10	Biokimia Lingkungan	ABS-47
Isolation and Characterization of Biosurfactants from Bioconversion of Palm Kernel Oil by Enterobacter Orizae and Bacillus Sp Vicky Rifatul Azkha , Rukman Hertadi Chemistry, Faculty of Science and Mathematic, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia Abstract Surfactant is a compound that can reduce the surface tension between two phases or the interfacial tension of a liquid such as water and oil. Surfactants are essential for the industrial production of detergents, textiles, paints, polymers, pharmaceuticals, pesticides, paper, and personal care products. For example, benzalkonium chloride and alkylbenzene linear sulfonate are employed to produce laundry detergents, personal care, and textile softeners. However, synthetic surfactants have several disadvantages for the environment because surfactants are one of the most difficult contaminants to remove and are continuously discharged into the environment through wastewater treatment plants (WWTP). Therefore, we can use biosurfactants as a substitute for chemical surfactants. Biosurfactants provide several advantages over surfactants derived from chemicals, especially in biodegradability, environmental compatibility, low toxicity, high selectivity, and specific activity at extreme temperatures, pH, and salinity. These biomolecules are produced by a diverse group of microorganisms, including fungi, bacteria, and yeast. The production of biosurfactants is significantly influenced by the type of substrate, fermentation technology, and microbial strains. Biosurfactants can be categorized as good if they can reduce the surface tension from 72 mN to 22 mN. This study assesses the potential of biosurfactants and determined the type of biosurfactants obtained from the isolation of bacteria residing in the gut of Indian termites, a collection from the Department of Microbiology, Tamil Nadu Agricultural University, India. After going through the screening and biosurfactant activity tests, two types of bacteria were found that can be used as biosurfactants. After going through the screening and biosurfactant activity tests, two types of bacteria were found that can be used as biosurfactants. The two types of bacteria are Enterobacter oryzae and Bacillus sp. The biosurfactant test using the Oil Spreading Test (OST) on Enterobacter orizae produced a clear zone diameters of 3.8 cm and 2.8 cm for Bacillus sp. In previous studies, it was verified that Bacillus sp could produce surfactant-type biosurfactants, but for Enterobacter orizae, there was no research as a producer of biosurfactants. Furthermore, optimizing the levels of NaCl, Nitrogen, and Palm Kernel Oil content will be carried out to optimize the production of the resulting biosurfactants and a specific physicochemical activity test will be carried out Keywords: Surfactant, Biosurfactant, Enterobacter oryzae, Bacillus sp, Oil Spreading Test (OST) Share Link | Plain Format | Corresponding Author (Vicky Rifatul Azkha)

11	Biokimia Lingkungan	ABS-48
cDNA from Total RNA of Navicula salinicola NLA Diatom Grown Indoor and Outdoor for sqRT-PCR Applications Trisha Audria, Sari Dewi Kurniasih Indrawan, Alfredo Kono, Yanti Rachmayanti, Zeily Nurachman* Biochemistry Division, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jalan Ganesa 10, Bandung 40132, Indonesia *zeily[at]itb.ac.id Abstract Navicula salinicola NLA, a potential biodiesel feedstock, shows light intensity-dependent lipid metabolism. However, knowledge about lipid metabolism pathways in N. salinicola NLA under different light conditions is lacking. This study aimed to obtain cDNA from N. salinicola NLA RNA for sqRT-PCR analysis. Methods included indoor/outdoor cultivation, total lipid isolation, primer design for internal gene control (rbcL and ACTB), genomic DNA isolation, primer compatibility testing, total RNA isolation, cDNA synthesis, and evaluation of control internal genes on N. salinicola NLA cDNA. The results shows that N. salinicola NLA cells had boat-like shapes, with dark brown (indoor) and light yellowish brown (outdoor) cultures. Initial cell density was 0.75 x 10^6 cells mL^-1, increasing to 4.1 x 10^6 cells mL^-1 (indoors) and 2.2 x 10^6 cells mL^-1 (outdoors) after 6 days. This resulted in specific growth rates of 0.33 +- 0.02 and 0.24 +- 0.03 x 10^4 cells mL^-1 day^-1, doubling times of 2.11 +- 0.12 and 2.94 +- 0.47 days, biomass productivity of 0.53 +- 0.01 and 0.23 +- 0.01 mg mL^-1 day^-1, and yields of 0.56 +- 0.08 and 0.41 +- 0.04 g L^-1 on the sixth day. Indoor cultures yielded lipids at 41.12%, while outdoor cultures yielded 46.1%. Internal control genes (NAVsa-RbcL-1 and NAVsa-ACTB-1) were identified from a 500 bp segment, demonstrating primer suitability. Total RNA was recovered during exponential and stationary phases, showing bands at 1400, 900, and 250 bp (indicating 28S, 18S, and 5S RNA) with a concentration range of 800-100 ng L^-1. ACTB was consistently expressed in both indoor and outdoor cultures. Keywords: Navicula salinicola NLA, Diatom, Triacylglycerol, Lipid, RNA, cDNA Share Link | Plain Format | Corresponding Author (Trisha Audria)

12	Biokimia Medis	ABS-1
Production of soluble single chain variable fragment (scFv) against Chikungunya virus in Escherichia coli Origami B (DE3) and its application as bioreceptor in immunosensor Shabarni Gaffar(1,2,3*), Siti Hesti Nurbayanti(1), Yeni Wahyuni Hartati(1), Mia Tria Novianti(2), Korry Novitriani(4), Safri Ishmayana(1), Muhammad Yusuf(1,3), Toto Subroto(1) (1)Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung, Indonesia (2)Research Center of Molecular Biology and Bioinformatic, Universitas Padjadjaran, Bandung, Indonesia (3)Graduate School, Universitas Padjadjaran, Bandung, Indonesia (4)Departement of Medical Laboratory Technology, Universitas Bakti Tunas Husada, Tasikmalaya, Indonesia. Abstract Chikungunya is an infectious disease caused by the chikungunya virus (CHIKV) with general symptoms of fever, skin rash, and joint pain that can last a long time. The available method for CHIKV detection is immunochromatography by using monoclonal antibodies to detect the E2 surface protein, since it was known to play a role in the attachment of CHIKV to the host cells. As a substitute for monoclonal antibodies, Single Chain Variable Fragment (scFv) can be used, which is part of the antibody that still has binding sites for E2. This study aimed to express anti-CHIKV E2 scFv in the Escherichia coli expression system and its application as bioreceptor in electrochemical immunosensor. The E. coli Origami B host cells carrying the pET21B-scFv plasmids were used to express scFv under control of the T7 promoter, and purified using a NiNTA column. Then, the scFv was immobilized on the surface of the gold modified Screen Printed Carbon Electrode (SPCE) and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that scFv was successfully expressed by obtaining a band of 30 kDa. Comparison of the expression results showed that co-expression of chaperone increased the soluble protein yield from 54.405 ug/mL to 220.097 ug/mL (5x). Furthermore, scFv can be used to detect CHIKV E2 in immunosensor electrochemistry with the optimum condition: scFv concentration (5.5 ug/ml), scFv immobilization (15 minutes) and CHIKV E2 incubation (45 minutes). The detection limit using the standard CHIKV E2 is 0.74048 ng/mL and the quantification limit of 2,24388 ng/mL. Thus, the scFv anti CHIKV E2 has the potential to be applied as a bioreceptor in another immunoassay method such as rapid test and ELISA. Keywords: Chikungunya, E. coli Origami B, soluble scFv, immunosensor electrochemistry. Share Link | Plain Format | Corresponding Author (shabarni gaffar)

13	Biokimia Medis	ABS-2
The D-enantiomer of LL-37 shows comparable antiviral activity against Zika virus as the native form of LL-37 Amalina Ghaisani Komarudin(a), Justus Obiajulu Sievers(a), R. Tedjo Sasmono(a), Anom Bowolaksono(b), Habibburahman Zulfikri(c), Pavel Svoboda(d), Jan Pohl(d), Craig Stevens(e), Peter G Barlow(e), Filipa Henderson Sousa(e)(f) a) Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Cibinong Science Center, Cibinong, Kabupaten Bogor 16911, Indonesia. b) Cellular and Molecular Mechanisms in Biological System (CEMBIOS) Research Group, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia c) Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok 16424, Indonesia d) Division of Scientific Resources, Centers for Disease Control and Prevention, Office of Technology and Innovation 1600 Clifton Road, NE Mailstop H21-8 Atlanta, Georgia e) School of Applied Sciences, Edinburgh Napier University, Sighthill Campus, Edinburgh EH11 4BN, United Kingdom f) Centre for Discovery Brain Sciences and UK Dementia Research Institute, The University of Edinburgh, Edinburgh EH16 4SB, United Kingdom Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus that is predominantly transmitted by Aedes mosquitoes. ZIKV can also be transmitted vertically from mother to fetus and by sexual transmission. There is currently no effective treatment for ZIKV infections, and given the spread of this disease worldwide, there is an urgent need for new, low-cost and efficacious treatments. LL-37, the sole human cathelicidin which are part of the innate immune system, has been shown to have antiviral activity against a wide range of enveloped viral pathogens. However, a major translational limitation of LL-37 is its susceptibility to post-translational modifications (PTMs) including proteolysis. In the present study, we investigated the in-vitro effect of the native form, D-form and scrambled LL-37 on ZIKV infection in A549 cells. The intracellular viral protein was quantified using the In-Cell Western assay. The culture supernatant was assessed for infectious viral particles by plaque forming unit assay and the level of cytokine release was determined using fluorescent microbead immunoassay. The L- and D-form of LL-37 effectively reduce ZIKV infectivity in A549 cells as compared to virus control. In terms of production of viral particles, the addition of D-LL-37 post infection showed infectivity reduction with a better significance than the native LL-37. Treatment of the ZIKV with scrambled LL-37 had no effect on ZIKV infectivity and the viral load as compared to virus control. Molecular docking studies revealed possible binding of L-form and D-form of LL-37 to the ZIKV envelope (E) protein dimer. The D-form of LL-37 binds to ZIKV E protein with a better stability compared to the native form. Together, our in-vitro experiments and in-silico analyses suggest that the native and D-form of LL-37 prevent the entry of ZIKV into the cells by binding to the E protein and engineering LL-37 into more biologically stable forms improve its antiviral potential against ZIKV infections. Keywords: ZIKV, antiviral, LL-37 Share Link | Plain Format | Corresponding Author (Amalina Ghaisani Komarudin)

14	Biokimia Medis	ABS-11
Biophysical characterization of inulin encapsulated insulin in simulated gastrointestinal fluid Achmad Ramadhannail Rasjava, Rukman Hertadi* Department of Chemistry, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia *rhertadi[at]itb.ac.id Abstract Development of biopolymer-based nanocarriers for oral insulin delivery has gained significant attention in recent years due to the challenges posed by insulin instability against gastric acid. This study evaluates the effectiveness of inulin produced by the halophilic bacterium Salinivibrio sp. GM01 as an insulin carrier. The synthesis of inulin-encapsulated insulin is optimized using the response surface method. The optimized conditions yielded a high entrapment efficiency of 87% by employing 53 mg of inulin, stirred at 17,800 rpm for 10 minutes. The resulting spherical nanoparticles exhibited an average diameter of approximately 511 nm. The efficacy of inulin as an insulin nanocarrier was assessed through encapsulation stability and conformational protection studies using simulated gastric and small intestinal fluids. Encapsulation stability remained consistent across both fluids, with inulin demonstrating resistance to pH variations. Circular dichroism and spectrofluorescence analysis revealed conformational stability differences between encapsulated and free insulin in gastric simulated fluid. Insulin encapsulation by inulin was shown to enhance the protection of insulin conformation by increasing the activation barrier for denaturation at the secondary and tertiary levels by 0.28 kcal/mole and 1.53 kcal/mole, respectively. These results highlight the potential of inulin as a promising material for the development of oral insulin delivery systems. The utilization of inulin-based nanocarriers offers a viable approach to address the challenges associated with insulin instability, thus paving the way for enhanced therapeutic outcomes in oral insulin delivery. Keywords: inulin-encapsulated insulin- conformational stability- insulin delivery- nanoparticles Share Link | Plain Format | Corresponding Author (Achmad Ramadhannail Rasjava)

15	Biokimia Medis	ABS-13
Antimicrobial Activity of Myrica javanica (Myricaceae) Extract Vienntiny Safrilianti Effendi1, Hany Pratiwi1, Yanti Rachmayanti2, Dikhi Firmansyah1 and Lia Dewi Juliawaty1* 1Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung, 40132, Indonesia 2Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung, 40132, Indonesia *Corresponding author, email: liadewi[at]itb.ac.id Abstract Myrica is a genus of plants belonging to Myricaceae family. It has been known to be used as a traditional medicinal plant for anti-asthma, skin diseases and stomachache. This genus contains cyclic diarylheptanoids as the main secondary metabolites that exhibit various bioactivities, such as anticancer, anti-inflammatory, antioxidant and antibacterial. One of the Myrica species growing in Indonesia is Myrica javanica in which the pharmacological studies are still limited. In this study, we investigated the antibacterial activity of acetone extracts of stem bark and twigs of M. javanica collected from the forest around Tangkuban Parahu Mountain, Lembang, West Java. Results showed that both extracts had significant antibacterial activity against food spoilage microorganisms, i.e., Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923, by Kirby-Bauer disc diffusion method. The diameter inhibition zones of stem bark and twigs extracts (2.5 mg) against Staphylococcus aureus were 22 mm and 21 mm, and against Pseudomonas aeruginosa were 24 mm and 19 mm, respectively. This study revealed that the acetone extracts of stem bark and twigs of M. javanica have potential antimicrobial activities. Keywords: Myrica javanica, stem bark, twigs, Pseudomonas aeruginosa, Staphylococcus aureus Share Link | Plain Format | Corresponding Author (Hany Pratiwi)

16	Biokimia Medis	ABS-17
Preliminary Study on the Purification of Anti-Envelope Protein Domain III (ED3) IgY Antibody for the DENV1 Virus using Silica Modified with Propyl Diethylenetriamine-Glutaraldehyde-ED3 Felicia (a), Yovin Sugijo (a), Fernita Puspasari (a), Ihsanawati (a), Wyanda Arnafia(c), Dessy Natalia (a), Handajaya Rusli (b), Fifi Fitriyah Masduki (a) (a) Biochemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia (b) Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia (c) PT Tekad Mandiri Citra, Jalan Raya Kawaluyaan No.20A Bandung 40286, Indonesia Abstract The WHO lists Indonesia as the country with the highest dengue hemorrhagic fever (DHF) cases in Southeast Asia. The cause of DHF is a Dengue virus infection that is transmitted to humans through the bite of the Aedes aegypti mosquito. High fever is the first symptom of the disease. To distinguish fevers caused by Dengue virus infection from other infections, the development of diagnostic tools that are accurate, fast, and economical is needed, such that dengue fever treatment can be carried out appropriately and quickly. One diagnosis method that meets the above requirements is Rapid Test Diagnosis (RDT) of antigens to diagnose Dengue virus in patient blood samples. One of the candidate biomarkers of the Dengue virus is blood Envelope Protein Domain III (ED3). A necessary component in the development of Dengue antigen RDT is the anti-ED3 antibody. Anti-ED3 antibodies in RDTs can interact specifically with viral antigens in blood samples so that dengue virus infection can be confirmed. Previous studies have successfully produced anti-ED3 IgY antibodies produced in chickens. To obtain anti-ED3 IgY antibodies that are usable in the development of Dengue antigen RDTs, it is necessary to carry out purification steps. Therefore, the purpose of this study was to purify anti-ED3 antibodies using propyl diethylenetriamine-glutaraldehyde-modified silica. The first stage of this research is to conduct an in silico study consisting of (1) analysis of amino acid side chains that can bind to glutaraldehyde and (2) analysis of epitopes on ED3 that can interact with anti-ED3 IgY antibodies using the ElliPro. The second stage in this study involves (1) the synthesis of propyl diethylenetriamine-glutaraldehyde modified silica using the Strober method, (2) expression of recombinant DENV1-ED3 protein, (3) immobilization of the ED3 protein on modified silica and (4) purification of the anti-ED3 antibodies. The results of the in silico studies showed that glutaraldehyde was reactive toward amino acid side chain residues containing secondary amino, and hydroxyl groups. Epitope prediction results show that epitopes exist on the surface of ED3 which can be easily accessed by anti-ED3 IgY antibodies. The success of modified silica synthesis can be seen from absorption bands in the FTIR spectrum, namely in 1109 cm-1 from Si-O-Si vibrations, 810-939 cm-1 from Si-C vibrations, and 1639 cm-1 from C=O vibrations. C-N bond vibrations at 136 cm-1 indicate that ED3 has been immobilized on modified silica. Furthermore, antibody purification was carried out using 0.5 M NaCl and 1 M NaCl as elution buffer. Although this anti-ED3 IgY antibody purification system still needs to be optimized, it has the potential to be further developed as a method for purifying other antibodies. Keywords: immobilization, silica, propyldiethylentriamine, glutaraldehyde, expression, ED3 protein, the anti-ED3 IgY antibody Share Link | Plain Format | Corresponding Author (Felicia Felicia)

17	Biokimia Medis	ABS-18
Nucleotide Sequence Determination of A Gene Encoding Sortase D from Rossellomorea aquimaris MKSC 6.2 Dewi Chairunnissa Wiyadi, Fernita Puspasari, Ihsanawati Institut Teknologi Bandung Abstract Surface proteins located on the cell walls of Gram-positive bacteria play an important role in the physiology and pathogenesis of these microbes. Sortase is a type of cysteine protease that helps the assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria. There are six classes of sortase, which has a different biological function, one of them is sortase D, involved in the spore formation and pili assembly. Sortase D in Bacillus anthracis is important for protein binding to the cell wall to facilitate sporulation, while sortase D in Bacillus cereus and other Gram-positive bacteria plays a role in pili assembly through the transpeptidase reaction mechanism. Current research on sortase D is still limited. Therefore, the aim of this study is to determine the nucleotide sequence of the gene encoding sortase D from the marine bacterium Rossellomorea aquimaris MKSC 6.2 (RaqsrtD). The study began with the design of degenerate primers based on information of the nucleotide sequence coding for class D sortases from various R. aquimaris strains in Genbank. Degenerate Polymerase Chain Reaction (degPCR) was performed to amplify the RaqsrtD gene fragment from the R. aquimaris MKSC 6.2 chromosome and it was inserted into pGEM-T Easy cloning vector. Escherichia coli TOP10F competent cells were transformed with this recombinant plasmid. The final step was determining the nucleotide sequence of the recombinant plasmid using the dideoxy Sanger method. Degenerate PCR produced a 260 bp gene fragment. The results of the transformation of E. coli TOP10F cells with pGEM-T-Easy-RaqsrtD260 produced 4 colonies that gave positive result in colony PCR analysis. Analysis of the nucleotide sequence of these 4 colonies using blastx on the NCBI website, showed that the size of RaqsrtD gene fragment is 263 bp and has similar nucleotide sequences (98.84%) to class D sortase from Bacillus sp. Marseille-Q1617 and Rossellomorea aquimaris CH159a_5T and CH87b_3T strains with percent identity 88.37%. These results underlied the preparation of primers to amplify the whole RaqsrtD gene (approx. 560 bp). Determination of the inserted gene sequence on the recombinant plasmid pGEM-T-Easy-RaqsrtD provided information that the size of the RaqsrtD gene that encode mature protein is 540 bp. The nucleotide sequence of RaqsrtD has percent identity of 99.47% with the nucleotide sequence coding sortase D from Bacillus sp. Marseille-Q1617. The study about determination of the nucleotide sequence of D sortase allows further characterization of this enzyme to develop drug target in preventing Gram-positive bacteria infection. Keywords: Cysteine protease, Sortase D, Rossellomorea aquimaris MKSC 6.2, degenerate PCR, nucleotide analysis Share Link | Plain Format | Corresponding Author (Dewi Chairunnissa Wiyadi)

18	Biokimia Medis	ABS-29
Molecular Modeling and Expression of Multimeric Bann-RBD Fusion Protein in Pichia pastoris as a COVID-19 Vaccine Candidate Please Just Ozi Jumadila (a), Muhammad Dzul Fakhri (a), Fernita Puspasari (a), Sari Dewi K. (a), Fifi Fitriyah M. (a), Keni Vidi Laseris (b), Ihsanawati (a), Dessy Natalia (a*)Try to Submit This Sample Abstract (a) Institut Teknologi Bandung, Indonesia (b) University of Helsinki, Finland *dessynatalia[at]itb.ac.id Abstract The COVID-19 pandemic caused by the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide impact on health, economy, and other sectors. Various vaccine platforms have been developed and are being administered worldwide to provide protection. Subunit protein vaccines are one of the most widely developed COVID-19 vaccine candidates. The receptor binding domain (RBD) of spike protein has been used as an antigen as it can stimulate the immune system to produce neutralizing antibodies that can inhibit the interaction of the virus with ACE2. Several studies have shown that dimeric, trimeric, and multimeric RBD produce higher neutralizing antibodies than monomeric RBD. In this study, we have developed a multimeric RBD as a protein fusion of RBD and &#946--annulus (Bann) from tomato bushy stunt virus (TBSV). Pichia pastoris X-33 was chosen as an expression host because it can secrete protein efficiently and has a post-translational modification system. The molecular modeling of the Bann-RBD protein shows a 60-mer structure with Bann being at the core and RBD being displayed on the surface. P. pastoris X-33-pPICZ&#945-A-Bann-RBD has been successfully constructed. SDS PAGE analysis of the Bann-RBD protein expressed in P. pastoris X-33 showed several protein bands which are predicted to be monomer, and multimer of glycosylated Bann-RBD. Further characterization indicated that the resulted recombinant Bann-RBD can recognize strongly an antibody of COVID-19 in positive patient serum. Taken together, this results indicate that the recombinant Bann-RBD produced in P. pastoris X-33 could be used in the development of COVID-19 candidate vaccine. Keywords: Keywords: COVID-19- multimeric protein- RBD- subunit vaccine- &#946--annulus Share Link | Plain Format | Corresponding Author (Ozi Jumadila)

19	Biokimia Medis	ABS-34
DNA barcodes for Identification of Indonesian Medicinal Plant Cryptocarya pulchrinervia Risma Septi Insani, Lia Dewi Juliawaty, Yanti Rachmayanti* Biochemistry Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia *rachmayanti[at]itb.ac.id Abstract Cryptocarya is a plant genus in Indonesia that has been studied for the activity of its secondary metabolites as anticancer, antiplasmodial, and analgesic agents. One species that contains secondary metabolites with cytotoxic activity, which can be developed as an anticancer agent, is Cryptocarya pulchrinervia. The aim of this research is to develop the DNA barcode for identification of the medicinal plant Cryptocarya pulchrinervia. The leaves of this plant, which have been studied for their secondary metabolites, were used as the source of DNA. The DNA isolation was carried out using Plant Genomic DNA Extraction from Viogene. 18S rDNA, rbcL, matK, and ITS marker genes were amplified by PCR technique. The isolated DNA and gene amplicon were confirmed by agarose gel electrophoresis and a nanodrop spectrophotometer. Several marker genes were sucessfully amplified, i.e 18S rDNA (ca. 250 bp), rbcL(ca. 1500 bp), matK (ca. 450 bp), and ITS (ca. 250 bp). These genes will be used for the sequencing analysis and the developing of DNA barcode for Cryptocarya pulchrinervia. Keywords: Cryptocarya pulchrinervia, marker genes, SNP, DNA barcode Share Link | Plain Format | Corresponding Author (Risma Septi Insani)

20	Biokimia Medis	ABS-39
Development Of Levan Nanoparticle System As A Material For Insulin Drug Delivery Desy Kurniawati(a*), Rukman Hertadi (a), Enny Ratnaningsih(a), Neng Fisheri (b) (a)Chemistry, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia *081383720011nanda[at]gmail.com (b)Pharmacy School, Bandung Institute of Technology Jalan Ganesha 10, Bandung 40132, Indonesia Abstract Levan is a biopolymer also known as fructan. It is a highly versatile material with various applications in industries such as medicine and chemistry. One of the uses of levan is in the field of nanotechnology, where it is used as a material to create nanoparticles. This study explores the potential of levan from the halophilic bacteria Bacillus licheniformis BK1 as a nanoparticle system for insulin delivery. FTIR was used to confirm the structure of levan. The obtained levan was acetylated and used as a nanoparticle system to immobilize insulin. The Insulin-levan nanoparticles (Lev-Ins) and Levan acetylation-insulin nanoparticles (Lac-Ins) have a size distribution in the range of 200-600 nm. Lev-Ins is stable at pH 4-7 but less stable at pH 1-3, while Lac-Ins is stable at various pH levels. The encapsulation efficiency of Lev-Ins and Lac-Ins was 78.46% and 88.30%, respectively. The results show that Lac-Ins can be used as a nanoparticle system for insulin delivery. Keywords: levan, nanoparticle, insulin Share Link | Plain Format | Corresponding Author (Desy Kurniawati)

21	Biokimia Pangan	ABS-30
The Effect of Adding Tempeh and Sodium Alginate on the Quality of Corn Milk (Zea mays L.) Angela Dwi Puspiturasia, Fida Madayanti Warganegara Department of Chemistry, Faculty of Mathematics and Science, Bandung Institute of Technology Abstract Corn milk is a plant based milk made from corn (Zea mays L.). Corn is a cereal crop rich in dietary fiber but has an incomplete amino acid profile compared to legumes. Therefore, this research aimed to vary the addition of tempeh to corn milk with concentrations of 0%, 5%, 10%, 15%, and 20% to increase the protein content of corn milk. The stability of the corn milk emulsion was analyzed by adding an edible film to obtain a stable milk emulsion using the settling rate order method. The shelf stability was determined by measuring the pH over a specific period to determine the length of time the corn milk remained suitable for consumption. Furthermore, the nutritional content of corn milk was analyzed, including protein content using the Kjeldahl method, carbohydrate content using the Luff Schoorl method, total dietary fiber using enzymatic methods, and fat content using gravimetric methods. The active compound beta carotene was also extracted from corn milk and its wavelength was determined using a UV Vis spectrophotometer to indicate the presence or absence of the compound in the corn milk sample. Additionally, sensory evaluation was conducted to assess the preference of the community towards corn milk, considering color, aroma, texture, and taste. Based on the research results, the corn milk preferred by the community was the one with 15% tempeh addition and an optimal edible film composition of 0.05% sodium alginate and 3% glycerol, with a shelf life of 4.5 days at a temperature of 25 degrees Celsius. The protein, fat, carbohydrate, total dietary fiber, and fat quality levels were determined as 0.85%, 3.2647%, 49.5 mg/mL, and 26.55 mg/mL, respectively. The beta carotene activity test indicated the presence of beta carotene compound in corn milk. Keywords: corn, tempeh, milk, edible film, beta carotene Share Link | Plain Format | Corresponding Author (Angela Dwi Puspiturasia)

22	Biokimia Pangan	ABS-52
Immobilization and Performance Evaluation of alpha-Amylase BmaN2 on Silica Propyldiethylenetriamine-Glutaraldehyde Matrix Rizka Chofifah Ahmad (a), Ihsanawati (b), Handajaya Rusli (a*) (a) Analytical Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung, Indonesia (b) Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung, Indonesia *Corresponding author: handajaya[at]itb.ac.id Abstract alpha-Amylase (E.C.3.2.1.1) is an enzyme that works specifically to hydrolyze alpha-1,4-glycosidic bonds in starch into glucose, oligosaccharides, and maltotriosadextrin units, which are raw materials for various industries. Starch processing with alpha-amylase has the least negative effects when compared to the use of non-enzyme chemicals. However, the starch processing industry requires an abundant amount of &#945--amylase because it cannot be used repeatedly. Immobilization is one of the techniques that can be used to allow repeated use of the enzyme. In this study, alpha-amylase from Bacillus megaterium NL3 (BmaN2) was immobilized on propyldiethylentriamine-glutaraldehyde silica matrix. This study also aimed to evaluate the performance of immobilized BmaN2 using starch solution. The matrix to immobilize BmaN2 was based on silica synthesized from tetraethylorthosilicate (TEOS) precursor by Stober method. The synthesized silica was further modified using N-1-(3-trimethoxysilpropyl)diethylentriamine (N-TMSPen)-glutaraldehyde. The success of silica synthesis was determined by infrared (IR) spectroscopy, i.e. the presence of absorption at wave number 1093 cm-1 (Si-O-Si vibrations) indicates that silica has been successfully synthesized. Typical vibrations of N-H at 1400 cm-1 and 3421 cm-1 indicate the successful modification of silica has been modified with propyldiethylentriamine. The C=O vibration at 1654 cm-1 indicated the successful modification of silica with glutaraldehyde, while the successful immobilization of the BmaN2 enzyme was marked by the presence of C=N vibration at 1544 cm-1. In addition to the results of the IR spectrum, the success of silica modification is determined from the color change of the matrix from white to brownish yellow also gives an indication of the success of the modification carried out. Based on SEM images, the morphology of silica, propyldiethylentriamine silica, and propyldiethylentriamine-glutaraldehyde silica is spherical. The presence of nitrogen atoms detected in Energy Dispersive X-ray Spectroscopy (EDS) also support the data on the success of the modification carried out. Based on Scanning Electron Microscopy (SEM) images, the morphology of silica, propyldiethylentriamine silica, and propyldiethylentriamine-glutaraldehyde silica is spherical. Characterization with Particle Size Analyzer (PSA) gave the average size of propyldiethylentriamine silica as 1174.6 nm, propyldiethylentriamine-glutaraldehyde silica as 1551.9 nm, and immobilized BmaN2 enzyme as 1174.6, 1551.9, and 5312 nm, respectively. In determining the enzyme performance test, the optimum conditions for free BmaN2 were pH 6, 30 minutes reaction time, and reaction temperature of 30 oC. For the immobilized BmaN2, the optimum conditions were pH 6, 90 minutes reaction time, and reaction temperature of 60 oC. The optimum starch solution concentration for immobilized BmaN2 was 2% m/v and free BmaN2 was 1% m/v. The Michaelis Menten constant (KM) values for immobilized and free BmaN2 were 1.55 and 2.23, respectively. These results show that the affinity of the enzyme in immobilized BmaN2 towards starch solution is higher than that of free BmaN2. Regarding the stability of BmaN2, the immobilized BmaN2 still has activity after being used repeatedly ten times and after being stored for two months in the refrigerator (0 oC). The results of this study indicate that silica modified with propyldiethylentriamine-glutaraldehyde can be used to immobilize BmaN2 so that it has the potential to be applied to the industrial world. Keywords: alpha-Amylase, immobilization, silica, N-TMSPen, glutaraldehyde Share Link | Plain Format | Corresponding Author (Rizka Chofifah Ahmad)

23	Bioteknologi	ABS-7
Kynurenine/Tryptophan Ratio As Promised Metabolomic Biomarker in Tuberculosis Infection Fitri Fadhilah (a*) , Agnes Rengga Indrati (b,c), Sumartini Dewi (d) & Prayudi Santoso (e) a)Doctorate in Medicine Program, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia. *fitrifadhilahssimkes[at]gmail.com b)TB Working Group, Infectious Disease Research Center, Faculty of Medicine, Universitas Padjadjaran, Jl. Prof Eykman, No. 38 Bandung 40161, Indonesia c)Department of Clinical Pathology, Faculty of Medicine, Universitas Padjadjaran/Hasan Sadikin General Hospital, Jl Pasteur No. 38 Bandung 40161, Indonesia d)Immunology Study Center, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia Division of Rheumatology, Department of Internal Medicine, Hasan Sadikin General Hospital, Bandung, Indonesia e)Respirology and Critical Illness Division, Department of Internal Medicine, Faculty of Medicine, Universitas Padjadjaran / Dr. Hasan Sadikin General Hospital, Bandung, Indonesia Abstract Immunology and the metabolic system have always been viewed as distinct disciplines. Recent advances in the understanding of immune functions under normal and disease conditions, have linked these disciplines to complex systems. Metabolomics, which is defined as the comprehensive evaluation of small molecule intermediates of metabolism within a biological system that collectively form the metabolome, has developed into an efficient method for discovering potential disruptions of metabolic homeostasis caused by disease. Recently, a prognostic metabolic biomarker with sufficient predictive power for tuberculosis progression has been developed. Targeted research of tryptophan in tuberculosis disease suggests such declines may also replicate induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting host enzyme for catabolism of tryptophan to kynurenine, which is highly upregulated in the lungs of tuberculosis disease patients. Although the latest diagnostic methods using tuberculosis nucleic acid amplification and interferon &#947- release assay have improved tuberculosis diagnosis, the lack of a biomarker for diagnosing active tuberculosis disease inhibits tuberculosis control. We hope that through this paper the reader can understand the benefits of metabolomics examination, especially as a type of examination that can be used to diagnose and monitor the effect of therapy in tuberculosis patients Keywords: infection- metabolomic- kynurenine- tryptophan- tuberculosis Share Link | Plain Format | Corresponding Author (Fitri Fadhilah)

24	Bioteknologi	ABS-12
Fabrication of Physically Crosslink Levan-lsbl-bk1/PVA Electrospun Nanofiber Bagus Furqan Abdillah (a), Muhamad Nasir (b), Tjandrawati Mozef (c), Rukman Hertadi (a*) a) Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl Ganesa no. 10 Bandung 40132, Indonesia *rhertadi[at]itb.ac.id b) Research Unit for Clean Technology, National Research and Innovation Agency, Jl. Sangkuriang, Kompleks LIPI Bandung 40132, Indonesia c) Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research and Innovation Agency, Jl PUSPITEK Serpong Gate, Muncul, Kec. Serpong, Tangerang Selatan 15314, Indonesia Abstract The failure to synthesise levan fiber with mechanical properties that match those of soft tissue requires an exploration into its fabrication. In this study, polyvinyl alcohol was used as a fiber-forming polymer for levan-lsbl-bk1 to produce fine electrospun fibers with the desired properties. The resulting fibers were physically crosslinked through heat treatment to improve their water resistance, and their chemical characteristics, crystallinity, morphology, and mechanical properties were identified. The diameters of the fibers ranged from 194.7-343.0 nm, with changes to the levan concentration affecting the fiber diameter and crystallinity. Furthermore, the tensile strength of the fibers decreased as the diameter increased, and the elastic modulus increased with increases in the crystallinity. The biocompatibility of the fibers was evaluated by analysing their effects on HepG2 cells since their elastic modulus of 12.7 MPa matched that of liver tissue. The use of the fiber resulted in an increase in HepG2 cell viability up to 158.8% on the first day and 215.5% on the second day compared to the controls. Keywords: PVA- levan lsbl-bk1 (Llb)- Electrospinning- HepG2 cell Share Link | Plain Format | Corresponding Author (Bagus Furqan Abdillah)

25	Bioteknologi	ABS-14
Production of poly-(R)-3-hydroxybutyrate (PHB) from halophilic bacterium Salinivibrio sp. utilizing palm oil mill effluent (POME) as a carbon source Wa Ode Sri Rizki, Enny Ratnaningsih, Rukman Hertadi Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha no. 10, Bandung, Indonesia, 40132 Abstract The accumulation of plastic waste has become an emerging threat to the ecosystem. Reducing plastic waste can be done by using biodegradable plastics, with polyR3hydroxybutyrate PHB as a potential candidate. Here, we successfully isolated, characterized, and optimized the PHB production of halophilic bacteria from salt lake Gili Meno, Lombok, Indonesia utilizing palm oil mill effluent (POME) as a carbon source. Isolate from the deepest part of the lake produced the most PHB of 5.00 plus minus 0.75 mg per L. Optimization of PHB production by this strain resulting of about 21.6 times higher production in the medium containing 15 percent of POME, 5 percent of NaCl, as well as 0.1 percent of yeast and 0.1 of ammonium sulphate as nitrogen source. The bacterium isolated was identified as Salinivibrio sp. with 99.9 percent similarity to the 16S rRNA gene of Salinivibrio sp. YCSC6 Genbank accesion no. CP039516.1. The infrared spectrum of the biosynthesized PHB having a maximum decomposition and degradation at 442.4 degree C and 282.9 degree C, respectively. This PHB was revealed to have a strong tendency to form a sheet-like structure. This research indicated that the isolated Salinivibrio sp. has a high potential in PHB production with high thermal stability by utilizing palm oil industry waste and makes it suitbale candidate to reduce the problem of plastic accumulation in the environment. In addition, the use of POME would also be economically beneficial as POME was abundantly available waste in palm oil industries. Keywords: poly-(R)-3-hydroxybutyrate (PHB), Salinivibrio sp., palm oil mill effluent (POME) Share Link | Plain Format | Corresponding Author (Wa Ode Sri Rizki Rizki)

26	Bioteknologi	ABS-21
Investigating the Role of Asp203 and His294 in the Catalytic Mechanism of BmaN1 Alpha Amylase Fina Khaerunnisa Frima (a,b*), Reza Aditama (a), Ihsanawati (a), Dessy Natalia (a) a) Chemistry Department, Institut Teknologi Bandung, Bandung, Indonesia. b) Chemistry Department, Institut Teknologi Sumatera, Lampung Selatan, Indonesia. Abstract BmaN1 is an alpha amylase produced by a sea anemone associated Bacillus megaterium NL3, which was isolated from from Kakaban Lake, East Kalimantan, Indonesia. Based on the similarity of amino acid sequences, BmaN1possesses aberrant catalytic residues within GH13 family and has recently been classified into the subfamily GH13_45. The amino acid sequence alignment and structure modelling of BmaN1, using alpha amylase from Geobacillus thermoleovorans (GTA, PDB code: 4E2O) as a template, revealed that only Glu231 was in the conserved position and functions as a proton donor. However, the other two of triad catalytic residues are different. Firstly, His294 functions as a transition state stabilizer in the conserved aspartate position. Additionally, Asp203 of BmaN1 is shifted to the i+1 position from the conserved nucleophile position, while Lys202 occupies the conserved nucleophile aspartate of GH13 alpha amylase. To investigate the role of catalytic residue Asp203 and His294, a truncated form of BmaN1 designated as BmaN1dC lacking 45 residues at the C-terminus was used. The aim of this research was to study the function of Asp203 and His294 in the catalytic mechanism. Site directed mutagenesis was performed to generate amino acid substitution Asp203Asn and His294Asp in BmaN1dC. BmaN1dC mutants was then produced in Escherichia coli ArcticExpress (DE3) and purified using Ni-NTA affinity column chromatography. Subsequently, the activity of BmaN1dC variants in starch hydrolysis was determined and it was found that BmaN1dC Asp203Asn and His294Asp lost up to 71% and 69% of their activity, respectively. These findings highlight the critical role of Asp203 and His294 in the catalytic mechanism of BmaN1. Keywords: alpha-amylase, BmaN1dC, GH13 family, catalytic residues. Share Link | Plain Format | Corresponding Author (Fina Khaerunnisa Frima)

27	Bioteknologi	ABS-22
Determination of Enzymatic Activity and Biophysical Characters of BmaN2, BmaN2 W198A, and BmaN2 W198F Alyn Shifa Aulya Wildania, Rindia Maharani Putri, Ihsanawati Institut Teknologi Bandung Abstract Starch, consisting of amylose and amylopectin, requires gelatinization for enzymatic hydrolysis due to its hydrophobic cavity that hinders water solubility. To reduce industrial costs, raw starch degrading amylase (RSDA) can be used, eliminating the need for gelatinization. BmaN2, an alpha-amylase produced by Bacillus megaterium NL3, lacks an extra starch binding domain but possesses starch-binding residues. Computational studies highlight Trp198 (W198) residue as potentially crucial for starch binding. Thus, this research aims to investigate the activity and biophysical properties of BmaN2 variants wild type, W198A mutant, and W198F mutant, regarding their interaction with raw and soluble starch. BmaN2 was obtained as a soluble protein by expressing it in E. coli BL21 (DE3), followed by purification using Ni-NTA affinity chromatography. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) confirmed successful BmaN2 expression, showing a band at approximately 61.5 kDa, corresponding to BmaN2^s size. Activity assays using soluble starch substrate revealed reduced activity in the pure mutant BmaN2, with a deficiency of 47% (W198A) and 48% (W198F) compared to the wild type. Spectrofluorometric analysis based on tryptophan emission indicated minimal structural changes among the three variants, as evidenced by the similar peak at 340 nm. However, fluorescence spectra demonstrated decreased emission intensity in BmaN2 W198A and W198F, suggesting increased tryptophan flexibility. The addition of starch solution caused a red-shift in wavelength from 340 nm to 355 nm in both the wild type and mutant BmaN2, indicating conformational changes due to starch interaction. This interaction opens the enzyme^s conformation, exposing tryptophan residues initially buried in the hydrophobic cavity. Circular Dichroism (CD) analysis revealed similar secondary structures between the wild type and W198F mutant, while the W198A mutant exhibited a distinct secondary structure. Keywords: alpha-amylase BmaN2, CD, raw starch, spectrofluorometric, substrate binding residues Share Link | Plain Format | Corresponding Author (Alyn Shifa Aulya Wildania)

28	Bioteknologi	ABS-24
Refolding of Recombinant Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor Binding Domain (RBD) Protein Using Fast Dilution Technique Hana Lalita Izdihar- Rindia Maharani Putri- Fernita Puspasari- Dessy Natalia- Ihsanawati Faculty of Mathematics and Natural Science, Bandung Institute of Technology Abstract The Receptor Binding Domain (RBD) of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS CoV 2) can recognize the human cell receptor, Angiotensin Converting Enzyme 2 (ACE2). This interaction is the initial step of virus infection in humans. Therefore, RBD is a protein target for therapy, vaccination, and studying the pathogenicity of SARS-CoV-2. Escherichia coli expression system is an ideal host to produce recombinant proteins because of its fast growth rate and ability to produce a large amount of protein. However, RBD production in E. coli results in inclusion bodies that affects the function and structure of RBD protein. Inclusion bodies are a consequence of protein misfolding and generally result in the loss of activity. The aim of this study is to refold the recombinant SARS-CoV-2 RBD protein structure using rapid dilution and characterize the RBD structure using spectrofluorometry. The first stage of the study was to transform E. coli BL21 (DE3) cells with a recombinant plasmid containing the RBD gene. The obtained transformants were induced with isopropyl &#946--D-1-thiogalactopyranoside (IPTG) to induce RBD protein expression. The RBD protein in inclusion bodies was dissolved in a buffer solution containing 8 M urea and purified by Ni-NTA affinity chromatography. The pure protein was then refolded using rapid dilution and characterized by spectrofluorometry. The transformed recombinant E. coli cells were able to grow in Luria Bertani (LB) medium containing ampicillin, and produced a band of 25 kDa on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This indicates that the cell transformation and RBD production stages were successfully conducted. The expressed protein was in the form of aggregates but was soluble in 8 M urea and produced a protein band of 50 kDa on SDS-PAGE. This result indicates the dimeric form of RBD in the denatured state. The 50 kDa protein band was more clearly visible after purification. The fluorescence spectrum after refolding showed a decrease in the percentage of fluorescence emission with decreasing urea concentration in the RBD solution. The normalized spectrum showed a maximum emission wavelength shift from red shift to blue shift. This wavelength shift is indicative of RBD conformational change from denatured to refolded state. Keywords: SARS-CoV-2, RBD, refolding, fast dilution, spectrofluorometry. Share Link | Plain Format | Corresponding Author (Hana Lalita)

29	Bioteknologi	ABS-25
Purification of Recombinant Nucleocapsid Protein of SARS-CoV-2 Yasmin Rizkya Putri, Ihsanawati Institut Teknologi Bandung Abstract Nucleocapsid protein or N protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the main antigen recognized by the human immune system. Therefore, N protein is an important target for developing diagnostic tests and anti-SARS-CoV-2 vaccines. The aim of this study was to produce and purify recombinant N protein of SARS-CoV-2. The research begun by transforming E. coli BL21 CodonPlus (DE3) RIPL cells with a recombinant plasmid containing the SARS-CoV-2 N coding gene so that these cells could grow in the antibiotic ampicillin medium. Successful transformation and production of protein N were confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The crude protein extract then subjected to purification via affinity chromatography and cation exchange. Purification of protein N using Ni-NTA affinity resin involves the coordination of histidine residues in protein N with Ni2+ ions present in the resin, forming covalent bonds. Protein N can be eluted from the resin using a buffer solution containing imidazole. For additional purification, cation exchange chromatography was employed, capitalizing on the isoelectric point (pI) of protein N, which is 10.07. By utilizing a Tris-HCl buffer with a pH of 8.0, protein N becomes positively charged. The electrostatic interactions between the functional groups on the resin and the protein can be disrupted by buffer solutions containing different concentrations of NaCl. The SDS-PAGE analysis of the crude protein N extract displayed a prominent band at approximately 45 kDa. However, purification using Ni-NTA resin resulted in closely spaced bands around 45 kDa, indicating the presence of protein impurities. Subsequent purification using HiTrap Capto S cation exchange resin revealed a single chromatogram peak with a concentration of 30 mAU, indicating the purity of protein N. The purified SARS-CoV-2 protein N can be utilized for future development of diagnostic kits. Keywords: COVID-19, SARS-CoV-2, NCoV2, E. coli BL21 CodonPlus (DE3) RIPL Share Link | Plain Format | Corresponding Author (Yasmin Rizkya Putri)

30	Bioteknologi	ABS-27
From sustainable cultures of Cyclotella striata diatoms to crystalline aluminosilicate reactors: a structural insight Rindia M. Putri,* [a] Novi Syahra Almunadya,[a] Amila Laelalugina,[a] Nadia Tuada Afnan,[a] Grandprix T.M. Kadja, [b,c,d] Rino R. Mukti,[b,d] Didin Mujahidin,[e] Yanti Rachmayanti,[a] and Zeily Nurachman* [a] [a] Dr. R. M. Putri, N. S. Almunadya, A. Laelalugina, N. T. Afnan, Dr. Y. Rachmayanti, and Prof. Dr. Z. Nurachman Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung 40132, Indonesia E-mail: rindia.m.putri[at]itb.ac.id- zeily[at]itb.ac.id [b] Dr. G. T. M. Kadja and Dr. rer. nat. R. R. Mukti Division of Inorganic and Physical Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung 40132, Indonesia [c] Dr. G. T. M. Kadja Center for Catalysis and Reaction Engineering, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, Indonesia [d] Dr. G. T. M. Kadja and Dr. rer. nat. R. R. Mukti Research Center for Nanosciences and Nanotechnology, Institut Teknologi Bandung, Jl. Ganesha No.10, Bandung 40132, Indonesia [e] Dr. D. Mujahidin Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung 40132, Indonesia Abstract Diatoms have emerged as a sustainable source of silica with unique structures and distinct pores. Compared to fossilized diatomites, diatoms live cultures can be grown continuously in a controlled manner, to result in a monodisperse and organized biosilica. Herein, we report the cultivation of a tropical marine diatom, Cyclotella striata, to produce amorphous biosilica with a large surface area filled by hierarchical pores. Via chemical incorporation of aluminium atoms into the Si-O-Si network, the micrometer sized biosilica was reassembled into smaller aluminosilicates with intergrowth structures, as corroborated by HR-TEM, SEM-EDX, and FTIR analyses. Furthermore, SAED and XRD revealed that the aluminosilicates displayed crystalline characteristics. The crystals were then employed as reactors to catalyze an etherification between ethanol and tertbutanol, to result in ethyl tert-butyl ether (i.e., an octane enhancer). Our findings demonstrate how sustainably grown diatom cultures can be structurally transformed into crystalline catalysts for the synthesis of high-value products Keywords: Microalgae, Diatom, Acid Catalyst, ETBE, Share Link | Plain Format | Corresponding Author (Nadia Tuada Afnan)

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