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:: Abstract List ::
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| 31 |
Bioteknologi |
ABS-28 |
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ROLE OF TRYPTOPHAN179 OF α--AMYLASE BMAN2 IN STARCH BINDING
Muhammad Aqib Hanif, Reza Aditama, Dessy Natalia
Institut Teknologi Bandung
Abstract
Plants typically use starch as the origin of carbon and energy. Seeds, roots, tubers, and legumes are the most common sources of starch. Enzymatic hydrolysis of raw starch can be accomplished with the aid of α--amylase. Bacillus megaterium NL3 from Lake Kakaban in East Kalimantan, Indonesia produces α--amylase BmaN2 which belongs to the GH13 family. BmaN2 has the ability to hydrolyse raw starch into linear and branched oligosaccharides. In Silico study of BmaN2 revealed that Tyr101, His141, and Trp179 are predicted to play role as SBS (Surface Binding Sites). The purpose of this research was to determine the role of W179 residue in substrate binding. Therefore, to find out the role of W179, two variants of bmaN2 constructed as bmaN2 W179F and bmaN2 W179A by site directed mutagenesis of TGG into TTC to produce bmaN2 W179F and TGG into GCG to construct bmaN2 W179A. Site directed mutagenesis was performed by PCR using a recombinant plasmid template pET30a-bmaN2 carrying bmaN2 gene. The expression of bmaN2 WT, bmaN2 W179F, and bmaN2 W179A in Escherichia coli BL21(DE3) was induced by the addition of 0.1 mM IPTG at 25 ℃- for 4 hours. SDS-PAGE analysis showed the presence of a protein with a molecular mass of 61.5 kDa. The specific activities for BmaN2 WT, BmaN2 W179F, and BmaN2 W179A were 1.61 U, 1.43 U, and 0.69 U, respectively. These results indicate that W179 of BmaN2 has an important role in substrate binding.
Keywords: α- -amylase, BmaN2, SBS, Site directed mutagenesis , tryptophan179
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| Corresponding Author (Muhammad Aqib Hanif)
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| 32 |
Bioteknologi |
ABS-31 |
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Expression and Characterization of Bann-RBD SARS-CoV-2 Delta in Pichia pastoris
Muhammad Dzul Fakhri, Ozi Jumadilla, Fernita Puspasari, Fifi Fitriyah Masduki, Ihsanawati, Dessy Natalia
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung, Indonesia
Abstract
The recent COVID-19 disease is caused by the highly infectious pathogen SARS-CoV-2, thus the development of vaccine is urgently needed in order to prevent the spread of disease. Protein subunit vaccine based on RBD of the viral spike protein is one of the leading platform in vaccine development for COVID-19. Several studies have shown that macromolecular structure, such as multimeric protein, can induce higher protective immunity against pathogen which can counter low immunogenicity of protein subunit vaccine.. Herein, we reported the expression and characterization of multimeric protein Bann-RBD in yeast Pichia pastoris X-33. The protein is a fusion of β--annulus (Bann) peptide from tomato busy stunt virus capsid and receptor binding domain of SARS-CoV-2 Delta variant. The construct encoding the whole Bann-RBD protein is integrated into Pichia pastoris X-33 chromosome via homologous recombination in the AOX1 promotor locus. The presence of ~1.25 kb DNA band in colony PCR confirmed the insertion of Bann-RBD in chromosome. The ~2.10 kb DNA band corresponded to the intact native AOX1 gene, hence the recombinant yeast is able to utilize methanol as carbon source (Mut+ phenotype). In the expression cassette, the AOX1 promoter regulates the transcription of Bann-RBD gene under the presence of methanol. Furthermore, Bann-RBD protein has the α--mating factor signal sequence which instructs the protein to be processed in post-translational modification and secreted to the medium. The ~44 and ~72 kDa protein band in SDS-PAGE gel are the glycosylated Bann-RBD protein expressed under methanol induction. Then, deglycosylation using endoglycosidase H resulted in a ~25 kDa protein which is the theoretical molecular weight of Bann-RBD. The optimum expression conditions for Bann-RBD protein are 72 hours of 2% methanol induction with initial OD600 of 10. Interaction analysis with ELISA showed that antibodies against SARS-CoV-2 can recognize the protein. Therefore, the results of this study suggest that the Bann-RBD protein can be a potential vaccine candidate for prevention of COVID-19.
Keywords: SARS-CoV-2, RBD, beta-annulus, Pichia pastoris
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| Corresponding Author (Muhammad Dzul Fakhri)
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| 33 |
Bioteknologi |
ABS-32 |
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Optimization of Chemically Structural Refolding of Spike 1 Recombinant Protein from SARS-CoV-2
Alberta Michelle Widyadi, Fernita Puspasari, Dessy Natalia, Ihsanawati
a) Chemistry, Faculty of Mathematics and Natural Science, Bandung Institute of Technology. Jalan Ganesha 10, Bandung 40132, Indonesia.
Abstract
S1 is a subunit of spike protein from SARS-CoV-2. This subunit plays a critical role for receptor interaction with human ACE-2 and virus fusion membrane process. S1 recombinant protein expression in E. coli produce an inclusion body that indicates the lost of protein native structure. This study aims to determine the optimum additives in buffer to help stabilize the S1 refolded recombinant protein. To achieve this goal, first the competent cell of E. coli BL21 CodonPlus (DE3) RIPL is transformed with recombinant plasmid pET-16b that carries the S1 encoded gen. Then, the transformed cell is grown in LB media containing ampicillin and induced by IPTG to expressed the S1 recombinant protein. After that, the protein is isolated and solubilized using 7.3 M urea. The solubilized protein is purified with Ni-NTA affinity chromatography. Next, the purified protein is refolded with slow dilution method using three additives that were added to the refolding buffer, N-lauroylsarcosine, arginine, and sucrose. The refolded protein is also characterized with Circular Dichroism spectroscopy. A band with the size of ~75 kDa is observed in the SDS-PAGE electrophoregram lysis cell crude extract. This band correlates to size of the S1 recombinant protein from the amino acid composition. The same band size was observed in theelution part of SDS-PAGE electrophoregram purification process. Highest recovery percentage was obtained using sucrose as an additives in buffer with the number of 95,4%. Sucrose provides a preferential hydration mechanism for protein stabilization, thus protein is surrounded by more water. Circular dichroism spectra demonstrated that the refolding process have restore the structure of S1 recombinant protein to the native state.
Keywords: inclusion bodies, refolding, SARS-CoV-2 virus, spike 1 protein
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| Corresponding Author (Alberta Michelle Widyadi)
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| 34 |
Bioteknologi |
ABS-33 |
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Characterization of Novel Thermostable Lipase from Family I.5 expressed in Pichia pastoris: in vitro and in silico study
Dimas Frananta Simatupang [a][b], Made Puspasari Widhiastuty [a], Fida Madayanti Warganegara [a], Akhmaloka [a]
[a] Biochemistry Division, Department of Chemistry, Institut Teknologi Bandung
[b] Department of Chemical Engineering, Politeknik Teknologi Kimia Industri Medan
Abstract
Itb1.1 was recombinant thermostable lipases from bacteria expressed in P. pastoris GS115 host cell extracellularly in a previous study. This research aimed to characterize a lipase in pure enzyme based on hydrolysis and transesterification activity. All experiments were investigated through the colorimetric method with slight modification to obtain optimum assay. The typical characteristics of Itb1.1 lipase was obtained that it worked well under optimum assay using medium-long acyl chains (C10), at temperatures 85 0C and pH 9.5 in alkaline condition. Furthermore, Itb1.1 lipase could maintain activity up to 50% for 4 h and tolerant in the presence of various organic solvents. The addition of Mg2+, Ca2+, Ni2+ and Fe2+ ions could enhance the activity of Itb1.1 lipase meanwhile the addition of Zn2+ ions inhibited its activity. This phenomenon revealed that Itb1.1 lipase was a unique and thought to be a novel lipase from Family I.5. This lipase was also stable in various surfactants. Moreover, transesterification activity was assessed to the thermostable lipase and revealed that Itb1.1 preferred medium-long chain fatty acid ester (C14:0). The in silico results were in agreement and supported with in vitro data. These results suggested that this lipase was potential biocatalyst for biodiesel production and various biotechnology applications.
Keywords: thermostable, lipase, Pichia, characterization, hydrolysis, transesterification
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| Corresponding Author (Dimas Frananta Simatupang)
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| 35 |
Bioteknologi |
ABS-35 |
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Starch - Coumarin Composite Films with Microcrystalline Cellulose - Biopolymer Blends as Biodegradable Plastic Materials
Amaliya Sita Permatasari, Rachmawati Rachmawati*
Inorganic and Physical Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132, West Java, Indonesia
* Corresponding author: rachmawati[at]itb.ac.id
Abstract
This research is a reference for developing starch-based plastic composite materials with microcrystalline cellulose (MCC). The low mechanical properties of starch biofilms necessitate the formation of inclusion complexes and the addition of a compatible polymer, such as cellulose. In this research, starch inclusion complexes were formed with coumarin molecules. The micro size of cellulose was added to fill the film matrix. In addition, using coumarin molecules in composite films can be an alternative method of adding aroma to bioplastic. The mass ratio of the plastic components were varied to produce bioplastics with good physical, good homogeneity and visual properties. The resulting plastic were analyzed to study their mechanical properties (tensile strength, strain, and Young^s modulus). The biodegradability properties were tested against microorganisms. Based on the results of mechanical tests, it was found that the double plasticizers used were PEG 200:glycerol (1:1) with a concentration of 20% (w/w), stirring time of 30 minutes and sonication of 50 minutes. The supporting biopolymers used were xanthan gum, karaya gum, and poly(vinyl) alcohol. The biodegradation test showed that Aspergillus Niger could degrade the starch film composite well. In addition, the starch - coumarin/xanthan gum complex composite showed the highest resistance to samples containing Trichoderma sp, Rhizobium sp, Azotobacter sp, Actinomycetes sp, Aspergillus sp, Bacillus sp, Lactobacillus sp, Pseudomonas sp.
Keywords: Biodegradation, Composite Film, Microcrystalline Cellulose, Starch
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| Corresponding Author (Amaliya sita Permatasari)
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| 36 |
Bioteknologi |
ABS-38 |
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Isolation of Total RNA from Navicula salinicola NLA for Transkriptomic Applications
Pradina Sofianti, Lifana, Alfredo Kono, Zeily Nurachman, Yanti Rachmayanti*
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung
Jl Ganesha 10, Bandung, 40132, Indonesia
*corresponding author: rachmayanti[at]itb.ac.id
Abstract
Navicula salinicola NLA is a species of unicellular microalgae that belongs to the diatom class (Bacillariophyceae) which has potential for biodiesel due to its lipid content. Knowledge of lipid metabolism pathways can help in efforts to increase lipid production by cells. Cell lipid metabolism can be understood through the information stored in datasets of mRNAs or transcriptomes. The aim of this study was to obtain total RNA isolates from the diatom Navicula salinicola NLA which qualified for transcriptomic analysis. The research steps included identifying diatom samples, cultivating cell samples in modified seawater medium, constructing cell growth curves, harvesting cell biomass from cultures in the exponential and stationary phases, total lipid extraction and total RNA isolation from both logarithmic and stationary phase cell biomass and quality analysis of RNA isolates. The results showed that the sample cells were identical to Navicula salinicola NLA which was characterized by light brown cell color, boat-like cell shape and bilateral symmetry. Initial cell density was 5,5 x 10^5 cells mL-1, increasing to 3,1 x 10^7 cells mL-1 after 7 days of cultivation. The total lipid content of cell biomass was 14% (w/w) in the logarithmic phase and 28.5% (w/w) in the stationary phase. The total RNA was 396.12 microgram isolated from the logarithmic phase and 605.72 microgram from the stationary phase. The ratio of A260/A280 was greater than 2.0 indicating a high level of purity of RNA isolates. The results of agarose gel electrophoresis showed clear and distinctive bands for RNA isolates.
Keywords: Biodiesel, Navicula salinicola NLA, lipid, RNA, transcriptomics
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| Corresponding Author (Lifana -)
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| 37 |
Bioteknologi |
ABS-40 |
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Expression of the Non-structural Protein NSP10 and NSP16 of the SARS-CoV-2 Coronavirus in Eschericia coli
Ita Saemena, Sari Dewi Kurniasih, Alfredo Kono, Yanti Rachmayanti, Zeily Nurachman*
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung
Jl. Ganesha No. 10, Bandung - 40132, Indonesia
*Corresponding Author: zeily[at]itb.ac.id
Abstract
Coronavirus (SARS-CoV-2) is an RNA virus containing genes encoding 16 non-structural proteins (NSPs) that are very important for the virus life cycle. When this virus infects a host cell, the viral RNA must be protected from the host cell immune system. The NSP10 and NSP16 complexes act as methyltransferase enzymes in the coronavirus which are crucial for the virus replication process in the host cell. The potential of NSP10 and NSP16 to be used to protect RNA structures generally is not limited to the SARS-CoV-2 viral RNA. Therefore, this study aimed to clone and express NSP10 and NSP16 of SARS-CoV-2 coronavirus in Escherichia coli and to test their activity in increasing RNA stability. Research methodology includes assembly of synthetic NSP10 and NSP16 genes from sequences available in the NCBI database, constructing of recombinant NSP10 and NSP16 genes each with pET16b and pETDuet expression vectors, transfer of recombinant plasmids into E. coli BL21 and pLysS, production and purification of protein, and protein expression analysis by SDS-PAGE. The results showed that the recombinant NSP10-pET16b and NSP16-pET16b were overexpressed in E. coli BL21 host cells by induction of 1 mM IPTG. The two proteins have different expression levels at various temperatures, where NSP10 is better expressed at 18 C while NSP16 is at 37 C. Crude protein was successfully isolated from E. coli cells by a combination method of freeze-thaw lysis and sonication. Pure protein was obtained by purification using Ni-NTA matrix and elution using 450 mM imidazole, i.e. as much as 1.00 ug/uL for NSP10 and 2.01 ug/uL for NSP16. The study will continue on testing the activity of the NSP10-NSP16 protein complex as a methyltransferase for RNA.
Keywords: NSP10- NSP16- SARS-CoV-2- methyltransferase
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| Corresponding Author (Ita Saemena Manuhutu)
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| 38 |
Bioteknologi |
ABS-41 |
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Purification and Characterization of Recombinant Alpha Amylase BmaN1dC from Bacillus megaterium NL3
Indri Novia Madhani (a*), Fina Khaerunnisa Frima (a,b), Dessy Natalia (a)
a) Chemistry Department, Institut Teknologi Bandung, Bandung, Indonesia
b) Chemistry Department, Institut Teknologi Sumatera, Lampung Selatan, Indonesia
Abstract
Alpha Amylase (E.C 3.2.1.1) catalyzes the hydrolysis of internal alpha 1,4-glycosidic bond of polysaccharides. Alpha Amylases are used in various industrial purposes such as textiles, detergent, paper, food, and pharmaceutical. A bacterium associated with a sea anemone Bacillus megaterium NL3 from the land-locked marine Kakaban Lake, Derawan Island, East Kalimantan, produces a unique alpha amylase BmaN1. BmaN1 is the first alpha amylase known to have different catalytic residues among members of the GH13 family. BmaN1 was produced in Escherichia coli BL21(DE3) as an inactive inclusion body due to the presence of transmembrane helical region at C-terminal of BmaN1. The aims of this study were to express, purify, characterize of the truncated BmaN1 which has no transmembrane helical region at C-terminal. BmaN1dC was produced in E. coli ArcticExpress (DE3) as a soluble protein with molecular weight of 49 kDa based on SDS-PAGE analysis. BmaN1dC has been purified to homogeneity with 10-fold purification with specific activity of 605 U/mg. The purified of BmaN1dC has exhibits high catalytic efficiency on soluble starch with kcat/KM value of 4.1 mL mg-1s-1. BmaN1dC retained about 35% and 44% on the presence of 10 mM EDTA and SDS, respectively. Despite no additional starch-binding domain, BmaN1dC is able to hydrolyze various raw starches, such as corn and cassava, in which any type of starch granular able to digested by BmaN1dC. In conclusion, these results indicated that BmaN1dC is potential to be used in starch processing industry within the subfamily GH13_45.
Keywords: Bacillus megaterium NL3, alpha amylase BmaN1dC, GH13_45 subfamily, catalytic residues
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| Corresponding Author (Indri Novia Madhani)
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