• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.667-667
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Life Science
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• v.13 no.6
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• pp.871-878
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• 2003

A marine bacterium (strain BKl) that produces extracellular enzymes capable of decomposing complex polysac-charides, such as agar, chitin, carboxymethylcellulose, xylan and mannan, was isolated from the marine red alga Porphyra dentata. Strain BKl was gram-negative, aerobic, catalase- and oxidase-positive, polarly flagellated bacilli that produce gelatinase and urease, but not decarboxylases. The G+C content of the DNA was 51.6 mol%. The major isoprenoid quinone component was identified as an ubiquinone-8, and the major cellular fatty acids were C16:0, C16:1 w6c and C18:1 w7c. Comparative 16S rRNA sequence analysis placed strain BK1 with members of the genus Pseudomonas. On the basis of phenotypic and genotypic data, the strain BK1 was shown to be a member of the subgroup of Pseudomonas, and named as Pseudomonas sp. BK1.

• KSBB Journal
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• v.29 no.4
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• pp.244-249
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• 2014

Biosynthesis pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHA) from fatty acid ${\beta}$-oxidation pathway was constructed in recombinant Escherichia coli by introducing the Pseudomonas sp. 61-3 PHA synthase gene (phaC2) and the maoC genes from Pseudomonas putida, Sinorhizobium meliloti, and Ralstonia eutropha. The metabolic link between fatty acid ${\beta}$-oxidation pathway and PHA biosynthesis pathway was constructed by MaoC, which is homologous to P. aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1). When the E. coli W3110 strains expressing the phaC2 gene and one of the maoC genes from P. putida, Sinorhizobium meliloti, and Ralstonia eutropha were cultured in LB medium containing 2 g/L of sodium decanoate as a carbon source, MCL-PHA that mainly consists of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), was produced. The monomer composition of PHA and PHA contents varied depending on MaoC employed for the production of PHA. The highest PHA content of 18.7 wt% was achieved in recombinant E. coli W3110 expressing the phaC2 gene and the P. putida maoC gene. These results suggest that MCL-PHA biosynthesis pathway can be constructed in recombinant E. coli strains from the b-oxidation pathway by employing MaoC able to supply (R)-3-hydroxyacyl-CoA, the substrate of PHA synthase.

• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.