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Stabilization of luciferase from Renilla reniformis using random mutations

Megumi Shigehisa, Norie Amaba, Shigeki Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, Fina Amreta Laksmi, Masao Tokunaga and Matsujiro Ishibashi

renillaWe expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli. RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

Keywords: Mutation, Stabilization, error-prone PCR, luciferase, Renilla reniformis

Katalog: http://perpus.biotek.lipi.go.id/index.php?p=fstream&fid=3143&bid=15591

Disusun oleh: Ludya AB/Pustakawan

 

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Aktivitas Antibakteri Aktinomisetes Laut Dari Pulau Enggano [Antibacterial activity of marine actinomycetes from Enggano Island]

Shanti Ratnakomala, Pamella Apriliana, Fahrurrozi Fahrurrozi, Puspita Lisdiyanti, Wien Kusharyoto

Berita Biologi, Volume 15 Nomor 3, Desember 2016, 275-283. ISSN 0126-1754

ntibacterial activity of marine actinomycetes from Enggano IslandMarine actinomycetes were isolated from mangrove sediment from the coast in Enggano, Bengkulu Province, Indonesia using the medium NBRC No. 802 modified by the addition of 2% NaCl. A total of 29 isolates of actinomycetes were isolated from three mangrove sediment samples and evaluated their potential to produce bioactive metabolites. Screening of 29 isolates marine actinomycetes isolates were performed against three bacterial pathogens had been done. Bacteria test used was Escherichia coli NBRC 14 237, Staphylococcus aureus NBRC13 276, and Bacillus subtilis NBRC 3134. Screening result showed that seven isolates have inhibitory effects against bacteria test and 22 other isolates have no inhibition. Of the seven isolates, one isolate has inhibitory effect against the growth of Gram-negative bacteria Escherichia coli, while six other isolates inhibit Gram-positive bacteria Bacillus subtilis and Staphilococcus aureus. It was concluded that, of the 29 isolates conducted in the experiment, seven isolates produce antibacterial compounds on agar medium. Molecular identification of 23 isolates were identified based on the gene 16S RNA sequences showed that 22 isolates belong to the genus Streptomyces and one strain belongs to the genus Dermacoccus.

Keywords: marine actinomycetes, antibacterial, Enggano Island

Katalog: http://perpus.biotek.lipi.go.id/index.php?p=fstream&fid=3371&bid=15841

Disusun oleh: Ludya AB/Pustakawan

 

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