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"The purification of recombinant proteins is an important stage in biopharmaceutical research. A commonly used technique is immobilized metal affinity chromatography (IMAC). One of the main advantages of this type of chromatography is that the column can easily be regenerated for subsequent purification work. The mechanism of IMAC is based on bonding between metal ions immobilized on a matrix with a specific amino acid. Because of the strong interactions of the electron donor group on the imidazole ring, histidine is often used in the IMAC purification system. Two types of commercial IMAC resin use a nitrilotriacetic acid (NTA) matrix: a nickel-based (Ni-NTA) and cobalt-based (Co-NTA), better known as TALON. This study was aim to investigate the effect of the metal ions Ni2+ and Co2+ to purify recombinant human erythropoietin (rhEPO) expressed in yeast system Pichia pastoris. The results indicated that both Ni-NTA and Co-TALON gave almost the same level of protein purity; however, Ni-NTA has a higher binding affinity than Co-TALON might be due to the higher stability complex of Ni+. The average amount of protein bound by Ni-NTA and Co-TALON was 183.5 and 38.7 μg/mL, respectively."

"Protein apoptin dari virus anemia ayam telah diteliti memiliki potensi yang baik sebagai pendeteksi dini sel kanker. Keberhasilan produksi apoptin yang tidak lagi berupa badan inklusi telah memberikan harapan lebih besar untuk melakukan optimasi produksi protein apoptin rekombinan ini. Sel rekombinan apoptin yang berhasil diproduksi dalam skala besar dengan menggunakan inang Bacillus subtilis 168 pOXGW-apop-2His8Arg, pOGW-apop-12His dalam berbagai variasi kondisi kultivasi (konsentrasi substrat penginduksi, laju aerasi, dan laju agitasi) kemudian dipurifikasi menggunakan metode IMAC dalam kolom afinitas (HisTrap FF 5 mL) yang berisi ion logam transisi Ni2+ dengan menggunakan instrumen AKTA Prime Plus. Secara rata-rata, hasil purifikasi menunjukkan bahwa elusi apoptin rekombinan terjadi ketika nilai konduktivitas berada pada angka 15,85 mS/cm, dengan konsentrasi imidazole berada pada kisaran nilai 76% - 88% (384,8-442,4 mM), yang terlihat dari grafik gradien elusi. Pengukuran konsentrasi protein apoptin dengan menggunakan metode Bradford menujukkan bahwa konsentrasi terbesar diperoleh pada sampel dengan sistem agitasi 250 rpm dan laju aerasi 0,5 Nl/menit, dengan besar konsentrasi 0,0507 mg/ml. Hasil purifikasi berhasil dideteksi menggunakan SDS-PAGE 12% dengan hasil pita protein terlihat di area 15 kDa dan 58,5 kDa untuk semua sampel.

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Apoptin has been known to be having a great potency for cancer detection. The success of apoptin production which is not in inclusion body form anymore has given a bigger hope to optimize its production. Recombinant apoptin cells which was succesful to be cultivated in large scale using Bacillus subtilis 168 pOXGW-apop-12His8Arg, pOGW-apop-12His vector in various cultivation condition (aeration rate, agitation rate) then purified using IMAC method in Ni2+-loaded affinity column (HisTrap FF 5ml) and proceeded in AKTA Prime Plus instrument. Averagely, purifcation result showed that the elution of apoptin recombinant protein happened when the conductivity value at 15,85 mS/cm, with imidazole concentration lied around 76%-88% (384,8-442,4 mM), which could be seen from elusion gradient curve. The measurement of apoptin protein concentration using Bradford method showed that the biggest concentration was obtained from the sample with agitation rate 250 rpm and aeration rate 0,5 Nl/min, and the value is 0,0507 mg/ml. Purification yield was succesfully detected using SDS-PAGE 12%, with protein band was seen on 15 kDa and 58,5 kDa area for all samples."

"Sistem CRISPR-Cas9 merupakan mekanisme perlindungan bakteri terhadap materi genetik asing yang diaplikasikan secara luas dalam rekayasa genetika. Kombinasi enzim Cas9 dengan gRNA pada CRISPR-Cas9 memungkinkan terjadinya pengeditan genom terhadap target yang spesifik. Meskipun demikian, Cas9 yang dikembangkan saat ini berasal dari bakteri mesofilik sehingga rentan terdegradasi dan tidak cocok untuk aplikasi pada temperatur tinggi. Di sisi lain, bakteri termofilik Geobacillus kaustophilus telah diisolasi dari mata air panas di Cisolong, Banten, dan diidentifikasi mengandung enzim Cas9. Untuk memperoleh enzim Cas9 yang tidak terdenaturasi pada temperatur tinggi (termostabil), dilakukan uji coba produksi Cas9 rekombinan dari Geobacillus kaustophilus. Gen Cas9 yang telah dikloning pada plasmid pET43.1a ditransformasikan ke dalam Escherichia coli BL21 dan dikultur dengan konsentrasi penambahan IPTG yang bervariasi—0,05 mM, 0,20 mM, dan 0,50 mM. Hasil kultur bakteri dipanaskan pada temperatur yang bervariasi (50 °C, 60 °C, dan 70 °C) untuk mendenaturasi enzim non-termofilik. Setelah itu, sampel dipurifikasi dengan mmobilized Metal Affinity Chromatography untuk memperoleh enzim Cas9. Hasil uji Lowry menunjukkan sampel heated supernatant dengan konsentrasi IPTG 0,05 mM dan temperatur pemanasan 50 °C memiliki konsentrasi protein tertinggi dan hasil purifikasinya memiliki konsentrasi Cas9 sebesar 42,6 μg/mL. Identifikasi protein dengan uji SDS-PAGE menunjukkan ukuran protein hasil purifikasi sebesar 52,61 kDa.

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The CRISPR-Cas9 system is a bacterial defense mechanism against foreign genetic material that is broadly applied in genetic engineering. The combination of Cas9 enzyme and gRNA in CRISPR-Cas9 allows genome editing of specific targets. However, the currently developed Cas9 originated from mesophilic bacteria, making it susceptible to degradation and unsuitable for applications requiring elevated temperatures. On the other hand, the thermophilic bacterium, Geobacillus kaustophilus, was isolated from a hot spring in Cisolong, Banten, and identified as containing the Cas9 enzyme. To obtain undenatured Cas9 enzymes at high temperatures (thermostable), a production test of recombinant Cas9 from Geobacillus kaustophilus was carried out. The Cas9 gene cloned on the pET43.1a plasmid was transformed into Escherichia coli BL21 and cultured under various IPTG addition concentrations—0.05 mM, 0.20 mM, and 0.50 mM. The bacterial cultures were heated at various temperatures (50 °C, 60 °C, and 70 °C) to denature unwanted non-thermophilic enzymes. Thereafter, the samples were purified using Immobilized Metal Affinity Chromatography to obtain Cas9 enzyme. Lowry protein assay results showed that the heated supernatant sample with 0.05 mM IPTG addition and 50 °C heating temperature has the highest protein concentration, and the purified sample yielded a Cas9 concentration of 42.6 μg/mL. Protein identification with SDS-PAGE revealed a purified protein size of 52.61 kDa"

"Perkembangan teknologi penyuntingan genom clustered regularly interspaced short palindromic repeat (CRISPR) memberikan kemampuan pada ilmuwan untuk memodifikasi sekuens genom pada sebagian besar sel eukariot. Selain untuk insersi dan delesi gen, penelitian sistem CRISPR-Cas9 juga telah menuju ke arah perkembangan represor transkripsional artifisial CRISPR interference (CRISPRi) dengan sistem dCas9 untuk rekayasa metabolisme melalui penyuntingan metabolic pathways.  dCas9 yang merupakan turunan dari Cas9 saat ini umumnya diproduksi oleh bakteri Streptococcus pyogenes yang merupakan bakteri mesofilik dan menyebabkan Cas9 dan dCas9 yang berasal dari Sterptococcus pyogenes memiliki limitasi terhadap suhu tinggi.  Saat ini eksplorasi terhadap bakteri termofilik sebagai sumber gen Cas9-dCas9 sedang berkembang. Salah satunya adalah bakteri Geobacillus kaustophilus yang tumbuh pada suhu optimal 60˚C dan dapat hidup hingga suhu 74˚C. Dalam penelitian ini, produksi enzim dCas9 dilakukan menggunakan Escherichia coli BL21 sebagai host dan dipurifikasi Immobilized metal affinity chromatography (IMAC). Variasi pemanasan supernatant dilakukan untuk suhu 50˚C, 60˚C, dan 70˚C sebelum purifikasi. Terdapat penurunan konsentrasi protein total dengan semakin tinggi suhu pemanasan, dengan konsentrasi protein total tertinggi pada suhu 50˚C. Purifikasi dilakukan menggunakan 3 buffer elusi dengan konsentrasi imidazole berbeda (250 mM, 350 mM, dan 450 mM). Konsentrasi imidazole 350 mM pada buffer elusi menghasilkan protein dengan konsentrasi total paling tinggi. SDS PAGE silver staining dilakukan untuk melihat berat molekul protein rekombinan yang telah dipurifikasi, dan protein terpurifikasi muncul pada pita ~50 kDa.

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The development of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing technology has given scientists the ability to modify the genome sequences of most eukaryotic cells. In addition to gene insertion and deletion, research on the CRISPR-Cas9 system has also led to the development of artificial transcriptional CRISPR interference repressors (CRISPRi) with the dCas9 system for metabolic engineering through editing of metabolic pathways. dCas9 which is a derivative of Cas9 is currently generally produced by Streptococcus pyogenes which is a mesophilic bacterium and causes Cas9 and dCas9 derived from Streptococcus pyogenes to have limitations against high temperatures. Currently, exploration of thermophilic bacteria as a source of Cas9-dCas9 genes is rising. One of them is the bacterium Geobacillus kaustophilus which grows at an optimal temperature of 60˚C and can live up to 74˚C. In this study dCas9 recombinant production was carried out using Escherichia coli BL21 as the host and Immobilized Metal Affinity Chromatography (IMAC) purification. Variation of supernatant heating was carried out for temperatures of 50˚C, 60 ˚C, and 70 ˚C before purification. There was a decrease in total protein concentration with higher heating temperature, with the highest total protein concentration at 50 ˚C. Purification was carried out using 3 elution buffers with varying imidazole concentrations (250 mM, 350 mM, and 450 mM). The imidazole concentration of 350 mM in the elution buffer produced fractions with the highest total protein concentration. SDS PAGE silver staining was performed to determine the molecular weight the purified fraction, and bands appeared in the ~50 kDa band."

"Penelitian karakterisasi produk gen sintetik lipase Thermomyces lanuginosus yang diekspresikan oleh Bacillus subtilis DB104 rekombinan K7 bertujuan untuk mengetahui pengaruh suhu, pH, dan ion logam terhadap aktivitas lipase. Bacillus subtilis DB104 promXynAQ1 non rekombinan digunakan sebagai kontrol. Lipase rekombinan optimal diproduksi pada media LB yang mengandung substrat minyak zaitun 1% selama 24 jam. Aktivitas lipase rekombinan diuji pada berbagai variasi perlakuan suhu (40°C--80°C), pH (5--10), dan penambahan ion logam menggunakan metode uji aktivitas spektrofotometri p-nitrofenil palmitat (pNPP assay). Data aktivitas spesifik lipase rekombinan dianalisis menggunakan data standar deviasi. Hasil penelitian menunjukkan bahwa lipase rekombinan aktif maksimal pada suhu 80°C dan optimal pH 8 dengan aktivitas spesifik sebesar 1,488 U/mg. Penambahan ion logam Ca2+, Mg2+, Cu2+, dan senyawa pengelat EDTA berpengaruh menghambat aktivitas enzim lipase rekombinan.

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The research of characterization of lipase Thermomyces lanuginosus synthetic gene product expressed by recombinant Bacillus subtilis DB104 had been conducted to investigate the effects of temperature, pH, and metal ions toward the enzymatic activity. Non recombinant lipase of Bacillus subtilis DB104 promXynAQ1 was used as control. Recombinant lipase was optimally produced using LB media containing 1% olive oil during 24 hours incubation time. Recombinant lipase was assayed in various treatments of temperature (40°C--80°C), pH (5--10), and metal ion addition using spectrophotometric method of p-nitrophenyl palmitate assay (pNPP assay). Specific activity of recombinant lipase data were analyzed with deviation standard. Experiment results showed that activity of recombinant lipase is maximum at temperature 80°C and optimum at pH 8 in the amount of 1,488 U/mg. The presence of metal cations Ca2+, Mg2+, Cu2+, and chelating-agent EDTA gave an inhibitory effect on recombinant lipase activity."

"ABSTRAKResistensi antibiotik merupakan salah satu masalah kesehatan yang membuat efektivitas pencegahan dan pengobatan berbagai infeksi menjadi berkurang. Peptida antimikroba seperti bakteriosin dapat menjadi kontributor penting dalam mengatasi permasalahan resistensi antibiotik sebagai agen antimikroba baru. Weissella confusa MBF8-1 diketahui menghasilkan tiga jenis bakteriosin yaitu Bac1, Bac2, dan Bac3 dengan sekuens DNA lengkap yang telah dilaporkan (A.N KR350502). Selain diproduksi melalui ekspresi galur produsen asalnya, bakteriosin dari W. confusa MBF8-1 sudah diproduksi melalui rekayasa genetika sehingga didapat bentuk rekombinannya pada inang B. subtilis DB403 dan disintesis secara kimia. Penelitian ini bertujuan untuk mengkarakterisasi aktivitas antimikroba serta efek sinergis dengan Ampisilin dari bakteriosin rekombinan dan sintetik Bac1, Bac2, dan Bac3 menggunakan metode difusi sumur agar pada Leuconostoc mesenteroides kemudian dilanjutkan dengan uji Konsetrasi Hambat Minimum (KHM) pada L. mesenteroides, Micrococcus luteus, Lactococcus lactis, Staphylococcus aureus, dan Escherichia coli. Hasil menunjukkan peptida sintetik memberikan zona hambat pada L.mesenteroides hanya dengan keberadaan Bac1. Peptida rekombinan tidak menunjukkan adanya inhibisi pada berbagai bentuk kombinasi. Peptida sintetik memiliki efek sinergis jika dikombinasikan dengan Ampisilin terhadap lima bakteri indikator, sedangkan peptida rekombinan tidak menunjukkan adanya efek sinergis. Efek sinergis terbaik yang teramati pada uji KHM dari peptida sintetik dicapai oleh Bac1, diikuti dengan bentuk campuran Bac1, Bac2, dan Bac3 (B1,2,3).

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ABSTRACTAntibiotic resistance is one of health problems that could decrease the effectiveness of infection treatment and prevention. Antimicrobial peptide like bacteriocin could be an important contributor to overcome antibiotic resistance as new antimicrobial agent. Weissella confusa MBF8-1 has been known to produce three types of bacteriocins, Bac1, Bac2, and Bac3, with their complete DNA sequences was reported previously (A.N KR350502). Besides produced through expression of origin producer strain, bacteriocin from W. confusa MBF8-1 has been produced through genetic engineering by recombinatorial process in B. subtilis DB403 and also by chemically synthesized. This study aimed to characterize the antimicrobial activity and the synergystic effect of those three recombinant peptides as well as their synthetic one with Ampicillin. Well-diffusion assay was performed using indicator bacteria Leuconostoc mesenteroides while Minimum Inhibitory Concentration (MIC) assay was performed using indicator bacteria L. mesenteroides, Micrococcus luteus, Lactococcus lactis, Staphylococcus aureus, and Escherichia coli. Result showed that synthetic peptides inhibited growth of L. mesenteroides when combined with Bac1. Recombinant peptides didn?t show any inhibition in various forms of combination. Synthetic peptides showed synergistic effect with Ampicilin against all indicator bacteria, while recombinant peptides showed no synergistic effect. The best synergistic effect with Ampicillin was showed by Bac1 synthetic peptide alone, followed by Bac1, Bac2, and Bac3 in combination (B1,2,3) by performing MIC test."

"ABSTRAKComplex air sample extracts were prefractionated on acidic- and basic-buffered silica. Compound overlap between acidic and basic polar substance (e.g. substituted polycylyc aromatic hydrocarbons) was greatly reduced, facilitating identification by gas chromatography-mass spectrometry. Furthermore, additional information on compound acidity and polarity is obtained, which helps to characterize compounds with identical electron-impact mass spectra but different structures."

"Indonesia mendeklarasikan target untuk mewujudkan net zero emission pada tahun 2060 mendatang. Banyak upaya yang telah dilakukan oleh pemerintah untuk mendorong ketercapaian target tersebut. Namun, beberapa sektor khususnya sektor industri masih menyumbangkan sebagian besar gas emisi-nya ke udara akibat dari limbah reaksi pembakaran dengan batubara. Amonia sebagai salah satu hydrogen carrier memiliki peluang dan potensi untuk dikembangkan menjadi solusi alternatif pengganti reduktor pembakaran batubara di sektor industri. Penelitian ini mengeksplorasi proses reduksi nikel laterit sintetik menggunakan gas amonia sebagai reduktor dan menganalisa efek variasi temperatur dan rasio reduktor terhadap fasa dan mikrostruktur. Nikel laterit sintetik diolah dari campuran oksida Fe2O3, NiO, SiO2, Al2O3, dan MgO dan dicampur dalam ball-milling yang setelahnya direduksi di dalam tube furnace. Penelitian ini menggunakan variasi temperatur di rentang 600-9000C serta rasio reduktor 1:1, 1:2, 1:3, dan 1:4. Waktu reduksi dilakukan selama 16-66 menit. Pengujian yang dilakukan diantaranya adalah XRD, OM, dan SEM-EDS. Hasil dari penelitian ini menunjukkan bahwa temperatur 9000C dengan rasio reduktor 1:4 merupakan kondisi yang optimal untuk mereduksi logam dari nikel laterit menggunakan reduktor amonia dengan persentase perolehan Fe sebesar 23% dan paduan FeNi sebesar 5%.

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Indonesia has declared a target to achieve net zero emissions by 2060. Many efforts have been made by the government to facilitate the achievement of this target. However, certain sectors, particularly the industrial sector, still contribute significantly to air emissions due to combustion waste reactions with coal. Ammonia, as a hydrogen carrier, has the opportunity and potential to be developed as an alternative solution to replace coal combustion reducers in the industrial sector. This research explores the synthetic reduction process of nickel laterite using ammonia gas as a reducer and analyzes the effects of temperature and reducer ratio variations on phase and microstructure. Synthetic nickel laterite is processed from a mixture of Fe2O3, NiO, SiO2, Al2O3, and MgO oxides, mixed in a ball-milling process, and subsequently reduced in a tube furnace. The study employs temperature variations ranging from 600-900°C and reducer ratios of 1:1, 1:2, 1:3, and 1:4. Reduction times range from 16 to 66 minutes. Tests conducted include XRD, OM, and SEM-EDS analyses. The results indicate that a temperature of 900°C with a reducer ratio of 1:4 is the optimal condition for reducing metals from nickel laterite using ammonia reducer, achieving a 23% yield of Fe and a 5% FeNi alloy."