PENGARUH MUTAGEN ULTRAVIOLET DAN ETIL METAN SULFONAT TERHADAP KADAR PENISILIN G PADA Penicillium chrysogenum

ABSTRACTPenicillium chrysogenum is one of the fungi which known to produce penicillin G antibiotics. The current superior strain P. chrysogenum producing high penicillin G has gone through various stages from the wild type isolates. The wild type isolates of P. chrysogenum from Indonesia also have great potential to be developed. Therefore, this study aimed to determine the changes of penicillin G levels from mutated P. chrysogenum isolate with both ultraviolet (UV) radiation and chemical mutagens ethyl methane sulfonate (EMS) compared to the wild type isolates. Random mutation through UV radiation, EMS chemical mutagen and their combination was carried out on wild type isolates of P. chrysogenum. Antibacterial activity was tested against Escherichia coli and Bacillus subtilis. The level of penicillin G produced was detected using HPLC with the Penicillin G as the standard. The results showed that the treatment of UV, EMS mutations and their combination can increase the antibacterial activity as well as levels of penicillin G than the wild type. Mutant C5-4.10 isolate resulted from the combination UV and EMS had the best antibacterial activity and produced penicillin G. level 2.9 times compared to the wild type.Keywords: antibiotic, HPLC, mutation, Penicillium chrysogenum, penicillin GABSTRAKPenicillium chrysogenum adalah salah satu kapang yang diketahui memproduksi antibiotik penisilin G. Strain P. chrysogenum unggul penghasil penisilin G tinggi yang saat ini tersedia merupakan galur yang telah melalui berbagai tahapan pemuliaan dari wild type-nya. Isolat P. chrysogenum asal Indonesia juga memiliki potensi besar untuk dikembangkan. Oleh karena itu, penelitian ini bertujuan untuk mengetahui perubahan kadar penisilin G dari isolat P. chrysogenum yang telah dimutasikan menggunakan radiasi sinar ultraviolet (UV) dan mutagen kimiawi etil metan sulfonat (EMS) dibandingkan dengan wild type nya. Mutasi acak menggunakan radiasi sinar UV, mutagen kimiawi EMS dan kombinasi keduanya dilakukan pada isolat P. chrysogenum. Uji aktivitas antibakteri dilakukan terhadap Escherichia coli dan Bacillus subtilis. Kadar penisilin G yang diproduksi dideteksi menggunakan HPLC dan dibandingkan dengan standar Penicillin G. Hasil penelitian menunjukkan bahwa mutasi menggunakan sinar UV, EMS dan kombinasi keduanya dapat meningkatkan aktivitas antibakteri serta kadar penisilin G dibandingkan dengan wild type nya. Isolat mutan C5-4.10 hasil mutasi kombinasi sinar UV dan EMS memiliki aktivitas antibakteri terbaik dan kadar penisilin G sebesar 2,9 kali lipat dibandingkan dengan wild type nya.Kata kunci: antibiotik, HPLC, mutasi, Penicillium chrysogenum, penisilin

Efektivitas Metode Ekstraksi DNA pada Daun Segar dan Kering dari Tanaman Obat

Tanaman obat merupakan salah satu tanaman yang sering menjadi target studi molekuler. Pada studi molekuler, ekstraksi dan purifikasi DNA genom merupakan tahap awal yang akan menentukan analisis selanjutnya. Oleh sebab itu, dibutuhkan metode ekstraksi DNA yang mampu menghasilkan DNA dengan kuantitas dan kualitas yang baik. Tujuan penelitian ini adalah untuk mengetahui efektivitas metode ekstraksi DNA dari daun segar maupun daun yang dikeringkan dari tanaman obat. Sebanyak sembilan genotip tanaman obat yang digunakan pada penelitian ini yaitu tiga genotip Justicia gendarussa, dua genotip Orthosiphon aristatus, dan masing-masing satu genotip Adenostemma lavenia, Adenostemma platyphyllum, Adenostemma madurense, dan Curcuma xanthorrhiza. Metode ekstraksi DNA genom yang digunakan adalah metode CTAB dengan beberapa modifikasi. Hasil penelitian DNA genom dari daun segar dan kering menunjukkan kuantitas, kualitas, dan dapat teramplifikasi dengan baik menggunakan primer RAPD dan penanda DNA

Development of Gold Nanoparticle (AuNP)-based Colorimetric Aptasensor for Penicillin G Detection

Antibiotics are chemical or biological substances that have the ability to kill pathogens selectively. Currently, high-performance liquid chromatography (HPLC) is used routinely in the detection of antibiotics. However, the cost of analysis and running time are bottlenecks for HPLC to be used for routine tests to detect antibiotics. Alternative methods need to be developed to overcome this issue. In this study, the development of a penicillin G specific biosensor by using a DNA aptamer and gold nanoparticles (AuNPs) was done. Optimal aptasensor conditions were achieved with the concentrations of NaCl and aptamer at 0.25 M and 2 μM, respectively. An aptasensor of this type showed LOD for penicillin G at 3 mg/L and was able to detect penicillin G in the range of 3 to 27 mg/L. The established aptasensor showed specific sensitivity toward penicillin G after testing with several antibiotics, i.e., ampicillin, kanamycin, chloramphenicol, and erythromycin. The aptasensor could detect the presence of penicillin G from culture medium of wild-type, ultraviolet irradiation mutant, gamma irradiation mutant, and ultraviolet irradiation and gamma irradiation mutant strains of P. chrysogenum, at detection concentrations of 9.75 ± 0.004; 25.25 ± 0.005; 37.5 ± 0.005; and 45 ± 0.004 mg/L, respectively

Development of Gold Nanoparticle (AuNP)-based Colorimetric Aptasensor for Penicillin G Detection

Antibiotics are chemical or biological substances that have the ability to kill pathogens selectively. Currently, high-performance liquid chromatography (HPLC) is used routinely in the detection of antibiotics. However, the cost of analysis and running time are bottlenecks for HPLC to be used for routine tests to detect antibiotics. Alternative methods need to be developed to overcome this issue. In this study, the development of a penicillin G specific biosensor by using a DNA aptamer and gold nanoparticles (AuNPs) was done. Optimal aptasensor conditions were achieved with the concentrations of NaCl and aptamer at 0.25 M and 2 μM, respectively. An aptasensor of this type showed LOD for penicillin G at 3 mg/L and was able to detect penicillin G in the range of 3 to 27 mg/L. The established aptasensor showed specific sensitivity toward penicillin G after testing with several antibiotics, i.e., ampicillin, kanamycin, chloramphenicol, and erythromycin. The aptasensor could detect the presence of penicillin G from culture medium of wild-type, ultraviolet irradiation mutant, gamma irradiation mutant, and ultraviolet irradiation and gamma irradiation mutant strains of P. chrysogenum, at detection concentrations of 9.75 ± 0.004; 25.25 ± 0.005; 37.5 ± 0.005; and 45 ± 0.004 mg/L, respectively

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3

Abstract Background A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. Results The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. Conclusion Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia