6 research outputs found
Isolasi dan Karakterisasi Molekuler Gen 16S rRNA Bakteri Lipolitik Asal Limbah Kulit Biji Jambu Mete: Isolation and Molecular Characterization of Lipolytic Bacterial 16S rRNA Gene from Cashew Nutshell Waste
No full textLipolytic bacteria attract great attention to various biotechnology industries because of their enzymatic potential. This study aims to isolate and identify lipolytic bacteria from cashew nutshell waste using the 16S rRNA gene as a molecular marker. Lipolytic bacteria were isolated using serial dilutions and inoculated on lipolytic media. A total of 3 isolates of lipolytic bacteria were obtained from cashew nutshell waste based on screening in LA Rhodamine B. The partial sequence of 16S rRNA gene from LB15 amplified using a pair of primers 63F and 1387R having a size of 1238 bp, while BL6 and BK6 were 1283 bp, respectively. Based on genetic distance analysis and phylogenetic reconstruction, we proposed that LB15 be identified as Burkholderia sp. with 99.92% similarity. In addition, because the 16S rRNA gene sequence similarity of BL6 was 99.87% with Paraburkholderia kururiensis strain 979, BL6 was classified as Paraburkholderia kururiensis. Then, isolate BK6 was identified as Ralstonia sp. with a similarity of 99.53%. The similarity value can be used as a reference in determining the identity of bacteria. A bacterium can be categorized as the same species if it has a similarity value of more than 99%.
Lipolytic bacteria attract great attention to various biotechnology industries because of their enzymatic potential. This study aims to isolate and identify lipolytic bacteria from cashew nutshell waste using the 16S rRNA gene as a molecular marker. Lipolytic bacteria were isolated using serial dilutions and inoculated on lipolytic media. A total of 3 isolates of lipolytic bacteria were obtained from cashew nutshell waste based on screening in LA Rhodamine B. The partial sequence of 16S rRNA gene from LB15 amplified using a pair of primers 63F and 1387R having a size of 1238 bp, while BL6 and BK6 were 1283 bp, respectively. Based on genetic distance analysis and phylogenetic reconstruction, we proposed that LB15 be identified as Burkholderia sp. with 99.92% similarity. In addition, because the 16S rRNA gene sequence similarity of BL6 was 99.87% with Paraburkholderia kururiensis strain 979, BL6 was classified as Paraburkholderia kururiensis. Then, isolate BK6 was identified as Ralstonia sp. with a similarity of 99.53%. The similarity value can be used as a reference in determining the identity of bacteria. A bacterium can be categorized as the same species if it has a similarity value of more than 99%.
 
Cloning and Co-Expression of Lipase and its Specific Foldase from Ralstonia pickettii BK6
No full textThis study aimed to obtain a functional lipase (LipRM) from Ralstonia pickettii BK6 through a co-expression involving its foldase. The ORF of the LipRM was 999 bp while the gene encoding of lipase-specific foldase (LifRM) was 1,030 bp. LipRM and LifRM genes were cloned into a plasmid and were successfully co-expressed in Escherichia coli strains to produce functional LipRM. Enzyme activity from partially purified enzymes showed quite surprising results, LipRM activity in E. coli BL21 (DE3) was 25.84 U/ml, while the other strains (DH5α, HB101, S17-1λpir) were 628.98 U/ml, 761 U/ml, and 1206.46 U/ml, respectively. The highest relative activity of LipRM was found at 50-55°C and pH 7-8 with pNP-laurate (C12) as the preferred substrate specificity. LipRM activity was enhanced sharply in the presence of 30% organic solvents (methanol and ethanol) but decreased by more than 50% in the presence of detergents. This study was the first to report heterologous expression of Ralstonia pickettii lipase employing its native foldase resulting in functional lipase from subfamily I.2
Analysis of the Molecular Structure of Lipase-Dependent Chaperone from Ralstonia pickettii Strain BK6
No full textSeveral biotechnology industries are exploring the characteristics of lipase-dependent chaperones due to their distinctive biochemical traits. This study aimed to employ bioinformatics to analyze the molecular structure of Ralstonia picketii BK6\u27s lipase-dependent chaperon (LipRM). The sequence mapping and amino acid distribution were examined using BioEdit (version 7.0.9.1). SignalP 5.0 and Interpro are employed for signal peptide detection, whereas Swiss-Model and VMD 1.9.2 are used for molecular dynamics modelling. The results showed that the Shine-Dalgarno sequence was discovered in the LipRM promoter, seven nucleotides upstream of the initiation codon (AUG) with the 5\u27-AGGAGA-3\u27, and has a terminator region that facilitates the formation of a secondary structure. The protein\u27s 3D structure prediction results indicate differences in the alpha helix chains (residues 166-174 and 254-271) between LipRM and the reference lipase. LipRM\u27s molecular structure comprises a detachable signal peptide, and with variations in helix alpha chain conformation and ligand geometry
Identifikasi Molekuler Bakteri Lipolitik Yang Diisolasi Dari Sedimen Mangrove Teluk Kendari: Molecular identification of lipolytic bacteria isolated from Kendari Bay mangrove sediments
No full textThe study aims to isolate and molecularly identify lipolytic bacteria from mangrove sediment in Kendari Bay, Southeast Sulawesi. Lipolytic bacteria were inoculated into Nutrient Agar Rhodamin B containing an olive oil emulsion and 2% NaCl by spread plate, then incubated at room temperature for 24-48 hours. Genomics DNA was extracted using the saline tris-edta (STE) method, while 16S rRNA gene was amplified using PCR technique involving universal primers 63F and 1387R. PCR conditions consisting of pre-denaturation (94ºC for 5 min), denaturation (95ºC for 30 sec), primer annealing (55ºC for 30 sec), extension (72ºC for 1 min), and post extension (72ºC for 7 min) were carried out 35 cycles. Bioinformatics analysis of 16S rRNA gene include similarity analysis using NCBI Blast, genetic distance matrix as well as phylogenetic tree reconstruction. The results revealed that two bacterial isolates (LMA1 and LMB3) exhibited lipolytic activity and had 16S rRNA gene sequence lengths of 1292 and 1283 bp, respectively. According to the results of BLAST analysis, the LMA1 isolate had a similarity value of 99.69% and a genetic distance of 0.0023 with Vibrio fluvialis, whereas LMB3 had a similarity value of 99.69% and a genetic distance of 0.0008 with Acinetobacter junii. This is also compatible with the phylogenetic tree classification of the two isolates. As a result, based on the partial 16S rRNA gene sequence, isolates LMA1 and LMB3 have the most genetic resemblance and/or are the same species as Vibrio fluvialis and Acinetobacter junii, respectively.
Key words: Lipolytic bacteria, 16S rRNA, mangrove sedimen
Analysis of the Molecular Structure of Lipase-Dependent Chaperone from Ralstonia pickettii Strain BK6
No full textSeveral biotechnology industries are exploring the characteristics of lipase-dependent chaperones due to their distinctive biochemical traits. This study aimed to employ bioinformatics to analyze the molecular structure of Ralstonia picketii BK6\u27s lipase-dependent chaperon (LipRM). The sequence mapping and amino acid distribution were examined using BioEdit (version 7.0.9.1). SignalP 5.0 and Interpro are employed for signal peptide detection, whereas Swiss-Model and VMD 1.9.2 are used for molecular dynamics modelling. The results showed that the Shine-Dalgarno sequence was discovered in the LipRM promoter, seven nucleotides upstream of the initiation codon (AUG) with the 5\u27-AGGAGA-3\u27, and has a terminator region that facilitates the formation of a secondary structure. The protein\u27s 3D structure prediction results indicate differences in the alpha helix chains (residues 166-174 and 254-271) between LipRM and the reference lipase. LipRM\u27s molecular structure comprises a detachable signal peptide, and with variations in helix alpha chain conformation and ligand geometry
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