In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²⁺ over Mg²⁺ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.

Penelitian ini dilakukan dengan tujuan untuk menentukan jenis mikroba koeksis denganascidia Lissoclinum patella menggunakan sekuens gen 16S rRNA. Sampel yang digunakandalam penelitian ini diambil dari jaringan tissue pada ascidia L. patella yang diambil dariperairan Malalayang, Sulawesi Utara. Sampel mikroba diinokulasi dalam media Hirata dandikultur selama ± 1 minggu. Sampel mikroba tersebut diisolasi DNA, amplifikasi melalui PCR(Polymerase Chain Reaction), elektroforesis gel agarose dan dianalisis data DNAnyamenggunakan BLAST pada NCBI (National Center for Biotechnology Information). Identifikasiyang dilakukan menggunakan BLAST diperoleh hasil 15 mikroba yang memiliki tingkatkemiripan yang tinggi dengan sekuens gen 16S rRNA sampel mikroba yaitu cyanobacteriumenrichment culture CAWBG121 dan CAWBG120 clone, uncultured Symploca sp. clone DRTO-55, Leptolyngbya sp. PCC7376 complete genome, Leptolyngbya sp. PCC7376, unculturedbacterium clone PINFEBB02, uncultured bacterium clone 5M47, Synechococcus elongatusCCMP1630, uncultured bacterium clone reef H09, Synechococcus sp. PCC7002 completegenome, Synechococcus sp. 16S rRNA gene strain PCC7002, Synechococcus sp. DNA untuk16 ribosomal RNA, Synechoccous sp. L21-BG-1, Oscillatoria rosea IAM M-220 danSynechococcus sp. PCC 8807. Tingkat kemiripan mikroba dalam NCBI dengan sampel H1berkisar antara 98-99 %.

Abstract. Wullur S, Napitupulu H, Wantania LL, Ginting EL, Warouw V, Tallei TE, Rumengan IFM. 2020. Molecular identification of bacteria isolated from culture medium of rotifer fed on fishery waste diet. Biodiversitas 21: 2735-2740. The aim of this study was 16S-rRNA sequences based molecular identification of bacteria isolated from culture medium of rotifer fed with fishery waste diet (FWD). We cultured rotifer Brachionus rotundiformis in sterilized seawater (salinity 25 ppt) using FWD, following the procedure in Patent No. P00201609066. Bacteria from the culture were collected, homogenized, diluted 10 to 1000 fold, spread on agar plates and incubated at 370C for 24 to 48 hours. Representative colonies of the bacteria according to their morphologies were isolated for further characterization. Genomic DNA of the isolates were extracted, and the 16S rRNA gene of the isolates were amplified. Polymerase Chain Reaction (PCR) product of each isolate was sequenced and queried against the NCBI GenBank database. Six different isolates based on size, color, elevation, margin, and colony were observed during 24-48 hours incubation at 370C. The 16S rRNA genes of the six isolates were successfully amplified and produced DNA band at 1300-1500 bp, with quality value equal to or greater than 20 (QV20+) of each entire sequence around 941-1253 bases. Basic Local Alignment Search Tool (BLAST) queries in the NCBI GenBank and EzBioCloud database using the 16S-rRNA gene sequences showed that the six isolates belong to four different genera, i.e: Bacillus, Staphylococcus, Vibrio, and Alteromonas.

The research was conducted at the Laboratory of Marine Molecular Biology and Pharmacy, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University. This research aims to isolate and analyze the morphology and molecular types of bacteria associated in rotifer’s culture media that use fisheries waste.The research was begin by culturing bacteria in rotifer maintenance media using Nutrient Broth media. After bacterial isolates were obtained, morphological characterization and DNA extraction was carried out. extraction was done using DNeasy Blood and Tissue Kit (Qiagen). After DNA was obtained, DNA was amplified through the Polymerase Reaction Chain (PCR) machine using a 16S RNA primer, followed by the separation of PCR products through electrophoresis and detection through UV Transluminator. The target PCR product was determined by comparing the 100 bp ladder DNA, with a yield of around 1400 bp, which was measured using ladder DNA available in the laboratory. The DNA that was successfully amplified was sent to be sequenced to determine the species of each microbe obtained.Based on the results of the research conducted, obtained Bacillus sp. bacteria associated with rotifer maintenance media.Keywords: Bacteria, Culture Media, DNA Extraction, PCR, Sequencing ABSTRAKPenelitian ini dilakukan di Laboratorium Biologi Molekuler dan Farmasetika Laut, Fakultas Perikanan dan Ilmu Kelautan, Universitas Sam Ratulangi.  Penelitian ini bertujuan untuk mengisolasi dan menganalisis morfologi dan molekuler jenis-jenis bakteri yang berasosiasi dalam media pemeliharaan rotifer yang menggunakan limbah perikanan.Penelitian dilakukan dengan cara mengkultur bakteri yang ada pada media pemeliharaan rotifer menggunakan media Nutrient Broth. Setelah isolat bakteri didapatkan, dilakukan karakterisasi morfologi dan dilakukan ekstraksi DNA.  ekstraksi dilakukan menggunakan DNeasy Blood and Tissue Kit (Qiagen).  Setelah DNA didapatkan, DNA diamplifikasi melalui mesin Polymerase Reaction Chain (PCR) menggunakan primer 16S RNA, diikuti dengan pemisahan produk PCR melalui electrophorisis dan deteksi melalui UV Transluminator. Produk PCR target ditentukan dengan membandingkan ladder DNA 100 bp, dengan hasil sekitar 1400 bp, yang diukur menggunakan ladder DNA yang tersedia di laboratorium.  DNA yang berhasil diamplifikasi, dikirim untuk dilakukan sekuensing untuk mengetahui spesies dari setiap mikroba yang didapatkan.  Berdasarkan hasil penelitian yang dilakukan, diperoleh bakteri Bacillus sp. yang berasosiasi pada media pemeliharaan rotifer. Kata Kunci: Bakteri,  Media Pemeliharaan, Ekstraksi DNA, PCR, Sekuensing