Judul: Heterologous Expression of Bacterial Lipases in Cereibacter sphaeroides 2.4.1
Abstrak: Penentuan sistem inang merupakan salah satu faktor yang penting untuk mengekspresikan protein rekombinan. Cereibacter sphaeroides 2.4.1 merupakan bakteri ungu non-sulfur yang memiliki potensi untuk dikembangkan sebagai sistem inang karena kemampuan metabolismenya yang sangat beragam. Bakteri ini dapat hidup pada kondisi aerobik maupun anaerobik sebagai organisme fototropik, kemotropik, autotropik, dan heterotropik. Kultivasi fotoheterotrof anaerobik adalah potensi dari bakteri ini karena dapat memanfaatkan energi cahaya tanpa perlunya penambahan oksigen kedalam kultur. Kondisi tumbuh ini menjadi penting untuk meningkatkan keberlanjutan pada industri bioteknologi. Penelitian bertujuan menguji C. sphaeroides 2.4.1 sebagai sistem inang untuk ekspresi heterolog lipase G. stearothermophilus T1.2RQ dan M. luteus EMP-48D. Gen lipase T1.2RQ dan EMP-48D diklon ke vektor ekspresi pRK415. Plasmid pRK415-T1.2RQ dan pRK415-EMP-48D kemudian ditransformasikan ke E. coli S17-1λpir. Koloni positif E. coli yang telah membawa plasmid rekombinan kemudian dikonjugasi dengan C. sphaeroides 2.4.1 menggunakan metode diparental mating. C. sphaeroides 2.4.1 yang telah membawa plasmid rekombinan kemudian digunakan untuk produksi lipase secara aerobik heterotrof dan anaerobik fotoheterotrof. Aktivitas lipase hanya terdeteksi pada C. sphaeroides 2.4.1 yang membawa lipase T1.2RQ. Lipase T1.2RQ diekspresikan secara intraseluler pada sistem inang C. sphaeroides 2.4.1. Aktivitas spesifik lipase T1.2RQ dari kultur aerobik heterotrof adalah 1,09±0,08 U/mg dan pada kondisi kultur anaerobik fotoheterotrof adalah 0,42±0,08 U/mg, sedangkan aktivitas spesifik pada kontrol E. coli BL21 bernilai 3,06±0,17 U/mg. Hasil ini menunjukkan aktivitas lipase T1.2RQ pada kondisi tumbuh aerobik 2,5 kali lebih tinggi dibandingkan anaerobik fotoheterotrof. Lipase T1.2RQ yang dihasilkan C. sphaeroides optimum pada suhu 65 ℃ namun hanya stabil hingga suhu 50℃ setelah inkubasi selama 180 menit. Suhu optimum yang diperoleh mengalami perbedaan dengan aktivitas lipase T1.2RQ yang diekspresikan pada G. stearothermophilus T1.2RQ dan E. coli. Lipase T1.2RQ optimum pada pH 10 dan stabil pada rentang pH 5-10, perubahan pH optimum juga terjadi pada lipase yang diekspresikan oleh C. sphaeroides 2.4.1. Lipase T1.2RQ diujikan pada beberapa substrat p-NP ester dan menunjukkan hasil tertinggi pada p-NP butirat, semakin pendek rantai asam lemak semakin tinggi aktivitas T1.2RQ. Ion Ca2+, Mn2+, dan Zn2+ mampu meningkatkan aktivitas lipase T1.2RQ yang dihasilkan C. sphaeroides. Lipase T1.2RQ dari C. sphaeroides memiliki ketahanan yang baik pada pelarut organik metanol, n-heksana, dan n-heptana. Penelitian awal ini menunjukkan bahwa ekspresi lipase rekombinan di C. sphaeroides 2.4.1 dapat dilakukan. Sistem ekspresi C. sphaeroides sebagai sistem inang perlu dikembangkan untuk meningkatkan hasil lipase rekombinan sehingga dapat diaplikasikan pada industri bioteknologi secara berkelanjutan.
Keyword: C. sphaeroides, Fotoheterotrof, Lipase T1.2RQ, pRK415

Judul: Extracellular Expression of thermostable Geobacillus stearothermophilus T1.2RQ Lipase for Biodiesel Production
Abstrak: The extracellular expression of enzymes in an excellent secretion host such as Bacillus subtilis is a useful strategy for making enzymes of industrial significance more affordable by lowering the cost of its downstream processing. Lipases are a category of hydrolases that catalyze the hydrolysis of triglycerides to glycerol and free fatty acids. Also, lipases catalyze the hydrolysis and transesterification of other esters as well as the synthesis of esters. They can perform very specific biotransformation reactions and are widely used in food, organic synthesis, detergents, cosmetic, and pharmaceutical industries. Geobacillus stearothermophilus T1.2RQ lipase is an industrially-desired thermostable lipolytic enzyme with an excellent hydrolytic and transesterification activity. This study aimed to express T1.2RQ lipase, in B. subtilis WB800 and ensure its secretion into the extracellular medium. Three signal peptides (AmyQ, Epr, LipA) and two promoters (Pgrac01 and Pgrac100) were used in the secretory-expression of T1.2RQ lipase, based on five constructed expression vectors constructed by restriction cloning and PCR mutagenesis. Sangers sequencing was used to verify constructs to ensure they are in the correct reading frame. Recombinants vectors, pHT43 (plasmid control), pHT43-T1.2RQ, pHT43- T1.2RQ_AmyQ, pHT43-T1.2RQ_Epr, and pHT43-T1.2RQ_LipA, were transformed by electroporation into Bacillus subtilis WB800. The electroporation was successful with the optimum condition of 2500V, 200 ohms, 25µF, and 5milliseconds. Recombinant B. subtilis pHT43 (negative control) and pHT43-T1.2RQlip + signal peptides were streaked on the lipidic agar, LA+TBN. All four recombinants, including the negative control (B. subtilis WB800-pHT43), produced clear zones on the TBN agar plate after overnight incubation (fig 2), indicating the presence of a lipolytic enzyme in all of the recombinant B. subtilis cells. pHT43-T1.2RQlip + signal peptides (AmyQ, Epr, and LipA) have clear zones with significantly higher diameters than B. subtilis WB800-pHT43 (negative control). This phenomenon suggests that the recombinant thermostable lipase T1.2RQ introduced into these cells was successfully expressed, thereby increasing the lipolytic capability of its host cells T1.2RQ lipase was expressed in 50ml terrific broth medium (TB) and the expression induced with 1mM IPTG. Lipase activity assay using p-nitrophenyl esters showed that all three signal peptides directed the secretion of T1.2RQ lipase into the extracellular medium. Signal peptide, LipA, resulted in the highest extracellular activity of 5.6 U/mL, which corresponds to a 6-fold increase over the parent B. subtilis WB800 strain. Meanwhile, signal peptides, AmyQ and Epr produced 4.2 U/mL and 3.4 U/mL activity respectively. SDS-Zymogram analysis confirmed that T1.2RQ lipase was correctly processed and secreted in its original size of 44 kDa. An attempt to further increase lipase expression level by promoter optimization showed that, contrary to expectation, promoter Pgrac100 exhibited similar expression levels as the original Pgrac01. Nevertheless, the demonstration of the successful secretion of T1.2RQ lipase in this work confirms the latter as a promising approach for the industrial production of the enzyme
Keyword: Bacillus subtilis, Expression, Extracellular, Heterologous, Lipase, Thermostable

Judul: Characteristic of Overshooting Top (OT) Based on  Himawari  8  Satellite  as  a  Convective  Cloud  Feature  that  Generates  Extreme Weather in the Java Island Region
Abstrak: Overshooting Top (OT) is a feature at the top of a convective cloud that is associated with extreme weather. Research on OT in tropical Indonesia is still very limited. The existence of geostationary satellites with wide area coverage and fast frequency observations is useful for observing the fast dynamics of the OT life cycle. Therefore, this study intends to analyze the relationship between OT and extreme weather, updraft and thermodynamic conditions of OT when extreme weather occurs, the best objective method for detecting OT in the day time and night time, and the spatiotemporal distribution of OT in the tropical region of Java Island during the 2018-2021 period. Based on visual analysis using visible channel and Himawari 8 sandwich products, 87% of extreme weather events in Java were accompanied by the appearance of OT. It indicates that the presence of the OT pattern is a convection cloud pattern or feature that is closely related to extreme weather events for the Java Island region. The existence of a time lag between the appearance of OT and extreme weather events on Java Island indicates that the OT of convection cloud can be used as a precursor or nowcasting for the appearance of extreme weather. Most of the convective cloud OT features during extreme weather events on Java Island have altitude that were only able to exceed the altitude of the equilibrium level and few were able to exceed the tropopause level of 9 km – 16 km. The difference in atmospheric conditions during extreme weather events on Java Island, which is in the tropics and subtropics, is a strong and unstable convective energy condition characterized by a larger CAPE, as well as unstable KI, LI, SI and TTI values. It is offset by kinematic factors in the form of a weak vertical wind shear (bulk wind shear 0-6 km) and a higher freezing point height. The difference between extreme weather events in the Java Island region and other tropical regions compared to subtropical regions is the strong updraft speed of convection clouds. The IRW Texture COMB algorithm based on the IRW-Texture Gradient modification and the COMB algorithm shows a significant increase in quality for detecting OT and non-OT pixels in Java. Rainy days with rainfall > 50 mm/day are very common in areas with an altitude of > 500 m. On the other hand, the level of convection activity which is indicated by the presence of OT and lightning is significantly reduced in this area. OT of convective clouds appear more frequently during the DJF season and less frequently during the JJA season. OT often appears during the day time and rarely at night time. Most OTs appear in the central part, away from the south and north coasts of Java Island during the day, while OTs that appear near the south and north coasts of Java Island can appear in the morning or afternoon.
Keyword: convective clouds, extreme weather, Overshooting Tops, Himawari 8 Satellite