• Korean Journal of Microbiology
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• v.49 no.2
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• pp.205-210
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• 2013

Lumazine protein is a fluorescent protein isolated from the bioluminescent bacteria of Photobacterium species. To generate minimal size of lumazine protein with possessing fluorescent characteristic, the gene coding for the wild type N-terminal domain of lumazine protein (N-LumP 118) containing amino acids up to 118 from Photobacterium leiognathi was produced. In addition, the genes coding for the variant proteins of N-LumP 118, replaced with one tryptophan amino acid (N-LumP 118 V41W, S48W, T50W, D64W, and A66W), were also constructed by Polymerase Chain Reaction and Site Directed Mutagenesis. These proteins were expressed in Escherichia coli by transformation with recombinant plasmids and purified by 6X-His tagging system. Spectroscopic studies have show that the purified proteins are capable of binding to the fluorescent ligand 6,7-dimethyl-8-ribityllumazine, resulted in showing of fluorescent characteristic with the minimal size of protein. From these studies, the mutant proteins containing single tryptophan amino acid residue, possessing its own intrinsic flouophore character at the different position, will be able to the use as a probe for further studies to deduce their three dimensional structure and the binding modes.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1708-1716
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• 2016

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BT^WT) and His-tagged (GDH-BT^N-His, GDH-BT^C-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BT^WT and GDH-BT^N-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT^C-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BT^WT was stable up to 65℃, whereas GDH-BT^N-His and GDH-BT^C-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BT^WT is a dimer, whereas GDH-BT^N-His and GDH-BT^C-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BT^WT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BT^WT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BT^WT and GDH-BT^N-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.1-10
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• 2007

The 44 endophytic bacterial strains were isolated from surface-sterilized root of rice cultivated in seven different locations of Chungcheong province, Korea. Each isolate was introduced into rice seedlings grown gnotobiotically by inoculating scissor-cut first true leaf with cell suspensions, and the colonization capacity of each isolate in root tissue was analyzed at 7 days after inoculation. Sixteen out of 44 isolates were re-isolated from root successfully with the frequency of $10^{3-5}$ CFU/g tissue. Interestingly, seven out of 16 isolates were identified as Burkholderia species. The identity between inoculated strains and re-isolates was confirmed by genomic finger-printing and 16S rDNA sequence analysis. By a confocal laser scanning microscopic observation it was revealed that KJ001 strain, one of the sixteen isolates tagged with gfp colonized in root tissue especially around xylem. Six out of seven Burkholderia strains obtained in this study showed antagonizing activities against seven different fungal pathogens, contain nifH gene, and five of them enhanced growth of cucumber over 30%. The isolates showed no hypersensitive response on tobacco leaves and no pathogenecity in rice. From these results it was found that the endophytic Burkholderia strains will be useful in agriculture to develop a biocontrol agent or a bio-fertilizer.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.318-326
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• 2002

The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia. To better understand this organism's strategies to survive osmotic stress, a mutant that was more sensitive to high osmolarity was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, putAP genes encoding a proline dehydrogenase and a proline permease were identified and cloned from V. vulnificus. The amino acid sequences deduced from nucleotide sequences of putAP from V. vulnificus were 38 to $59\%$ similar to those of PutA and PutP reported from other Enterobacteriaceae. Functions of putAP genes were assessed by the construction of mutants, whose putAP genes were inactivated by allelic exchanges. When proline as the sole carbon or nitrogen source was used, the putA mutant was not able to grow to the substantial level, revealing the proline dehydrogenase is the only enzyme for metabolic conversion of proline into other amino acids. Although the growth rate of the putP mutant on proline as the sole carbon or nitrogen source was significantly reduced, the mutant still grew. This indicated that at least one more proline permease is produced by V. vulnificus. The putP mutant decreased approximately $2-log_10$ CFU/ml after a hyperosmotic challenge, while the parent strain decreased approximately $l-log_10$ CFU/ml. This result suggests that the gene product of putP contributes to the osmotic tolerance of V. vulnificus.