In-Hye Park

Discovery of three novel lipase (lipA1, lipA2, and lipA3) and lipase-specific chaperone (lipB) genes present in Acinetobacter sp. DYL129

A microbe isolated from a soil sample obtained on Deog-yu Mountain in Korea, was found to produce an extracellular lipase. This microbe, which was designated as DYL129, was identified as an Acinetobacter sp. based on phylogenetic analysis of its 16S rDNA. A genomic library was constructed by using DYL129 fragment digested with HindIII and a recombinant plasmid, pLip-1, was selected for further analysis by colony polymerase chain reaction (PCR). Sequencing of a 3.8-kb insert in the pLip-1 clone revealed the presence of one incomplete and three complete open reading frames (ORFs). The ORFs were predicted to encode a partial lipase, two putative lipases and a 50S ribosomal protein. Genome-walking PCR also identified transcripts encoding a complete lipase chaperone and three lipaseA proteins. The lipase structural gene present in Acinetobacter sp. DYL129 was similar to the lipase structural gene found in Acinetobacter calcoaceticus BD413 (lipBA). However, the three lipase genes were located downstream of the chaperone gene in Acinetobacter sp. DYL129 (lipBA1A2A3), which differs from the location of these genes in A. calcoaceticus BD413. Although the amino acid sequences of these lipases (LipA1, LipA2, and LipA3) differed, both strains had a common “GHSHG” consensus motif, which is the conserved active-site pentapeptide of lipaseA. Moreover, all three lipases were found to share a conserved domain, the so-called α/β hydrolase fold.

Isolation, Purification and Characterization of the β-Xylosidase from Klebsiella sp. Sc.

A -xylosidase encoding gene from Klebsiella sp. Sc was cloned in Escherichia coli. The -xylosidase gene consisted of an open reading frame of 1,680 nucleotides and encodes 559 amino acids with a deduced molecular weight of 63 kDa. The deduced amino acid sequence of the -xylosidase from Klebsiella sp. Sc exhibits 90% identities and 95% positives compared to those from Klebsiella oxytoca (KOX), Lactobacillus lactis (LAC, 82%, 90%), Bacillus longum (BLON, 69%, 81%) and Escherichia coli (ECOLI, 47%, 63%). The -xylosidase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 6.6 and , respectively. The -xylosidase hydrolyzes xylobiose to xylose.

Molecular Cloning and Analysis of Phosphate Specific Transport (pst) Operon from Serratia marcescens KCTC 2172

A recombinant plasmid, pDH3, was obtained from the genomic library of Serattia marcescens KCTC 2172, and several recombinant subclones constructed from pDH3. The nucleotide sequence of a 5,137 bp segment, pPH4, was determined and three open reading frames were detected. The three ORFs encoded the phosphate specific transport (pst) operon, which was pstC, pstA, and pstB, with the same direction of transcription. Comparison of the pst operon of S. marcescens with that of other organisms revealed that the genes for pstS and phoU were missing. A potential CRP bonding site and pho box sequence was found in the upstream of the putative promoter at the regulatory region. Analysis of the nucleotide sequence showed that homology in amino acid sequences between the PstC protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. were 49, 37 and 33%, respectively. The PstA protein and Yersinia sp., Vibrio sp., and Pseudomonas sp. showed homologies of 64, 51, and 47%, respectively. PstB protein and Methanocaldococcus sp., E. coli, and Mycoplasma sp. showed homologies of 60, 50, and 48%, respectively. The pst genes could be expressed in vivo and positively regulated by cAMP-CRP. The E. coli strain harboring plasmid pPH7, with pst genes, increased with the transport of phosphate.

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.12 No.3, Issue of July 15, 2009

Nutritional Characteristics of Water Extract from Kangaroo Tail.

To get the nutritional data of hot water extract from imported kangaroo tails, research was done about contents of collagen, chondroitin sulfate, protein, fat, mineral ions, and conjugated linoleic acid (CLA) in tails. Collagen content of sulfuric acid digested sample was way higher at bones than meats in both kangaroo tail and cow tail. Comparing kangaroo tail and cow tail, meat of kangaroo tail have 1.7 times higher collagen content than that of cow tail. Content of collagen in bone parts of kangaroo tail was also higher 1.2 times than that of cow tail. Meat sample of kangaroo tail (liquid extraction) have 1.3 times higher content of muco-polysaccharide than that of cow tail. In the born part, kangaroo tail was 2.4 times higher than cow tail in its content of muco-polysaccharide. The CLA content of the kangaroo tail showed the content that was higher than 0.9% of the cow tail for 4.9% and showed about 5.3 times high ratios. Especially in kangaroo tail, a band with high content of CLA was found between C18:1 and C18:2.

Improvement of the Phosphate Solubilization Microorganism by the Introduction of Glucose Dehydrogenase Gene into Aeromonas hydrophila DA33.

Aeromonas hydrophila DA33 was isolated from cultivated soils as a bacteria having high abilities to solubilize inorganic phosphate. Glucose dehydrogenase gene (gdh) was cloned from Escherichia coli. The recombinant plasmid, pGHS containing glucose dehydrogenase gene was introduced into A. hydrophila DA33 in order to improve the activity of phosphate-solubilizing. The transformant harboring the gdh gene, A. hydrophila pGHS/DA33 increased enzyme activity. The strain also increased the gluconic acid generation that was effective for phosphate solubilization. It was possible that the strain containing pGHS produced higher solubilized phosphate with tri-calcium phosphate as the unique (P) source, in comparison with that of wild type without plasmid. These results suggest that the strain, A. hydrophila pGHS/DA33 is expected as effective biofertilizer for phosphate solubilization.

Isolation and Characterization of Acinetobacter sp. BD5 Producing Lipolytic Enzyme

A bacterium producing novel lipolytic enzyme was isolated from house sewage and identified as Acinetobacter sp. BD5 based on physiological characterization and 16S rDNA sequencing. The lipolytic activity of Acinetobacter sp. BD5 was tested using an EL agar medium and CE agar medium supplemented with 1% tributyrin and olive oil, respectively. The formation of a clear zone around the colony was detected by agar medium supplemented with 1% tributyrin and olive oil, respectively and Acinetobacter sp. BD5 formed powder-like zone around the colony on LB agar medium containing Tween 20. The quantitative lipolytic activity was determined by using p-NP butyrate as substrate. Acinetobacter sp. BD5 secreted the lipolytic enzyme during exponential growth phase, reaching a maximum amount after 6 hours of incubation. The lipolytic enzyme was found to be optimally active at and retained more than 70% at . It displayed a high degree of activity in a pH of 7.0 to 10.6, with an optimal pH of 9.0.