• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.315-320
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• 1996

The role of glyoxylate bypass in a lysine-producing Corynebacterium lactofermentum strain was analyzed. Unlike the wild type, the strain expressed enzymes of glyoxylate bypass during growth in the fermentation broth containing glucose as the carbon source. To evaluate the importance of glyoxylate bypass in the strain, we disrupted chromosomal aceA by using a cloned fragment of the gene. Site-specific disruption of aceA which codes for the isocitrate lyase, the first enzyme of the bypass, was confirmed by Southern blot analysis. The aceA mutant strain completely lost isocitrate lyase activity and ability to grow in a minimal medium containing acetate as the sole carbon source. The mutant strain was similar to its parental strain in growth characteristics and produced comparable amounts of lysine in shake flasks containing glucose as the carbon source. The amount of oxaloacetate accumulated in the fermentation medium was similar for both strains, suggesting that expression of glyoxylate bypass does not necessarily lead to the increase in intracellular oxaloacetate. These data clearly demonstrate that glyoxylate bypass does not function as one of the routes of carbon supply for lysine production in the strain. It appears that the leakiness of the glyoxylate bypass in the strain might be the result of a secondary mutation which arose during previous strain development by random mutagenesis.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.321-325
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• 1996

Mutants having altered levels and/or types of EPS in exopolysaccharide biosynthesis were isolated after NTG mutagenesis of Zoogloea ramigera 115SLR. Mutant candidates were classfied with five groups based on the observed characteristics for EPS biosynthesis pattern. The recombinant plasmids pLEX3BS and pLEX3BM were constructed from pEX3B which was previously isolated from genomic DNA of Z. ramigera 115SLR. Plasmid pLEX3BM with a 7.8 kb insert DNA contains an additional 1.8kb DNA fragment which is not present in pLEX3BS containing 13 kb insert DNA. Plasmid pLEX3BM was able to complement the mutation responsible for the changes in morphology of Z. ramigera 115SLR. However, the complementation of EPS negative mutant strains was not successful with pLEX3BM. Plasmid pLEX3BS on the other hand complemented the mutation responsible for the loss of EPS biosynthesis, resulting in the restoration of Z. ramigera 115SLR phenotype. But this plasmid was not able to complement the morphological mutant strain, Z. ramigera 115SLR.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.434-437
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• 2003

A new bacterial strain, was isolated from activated sludge, identified and named P. aeruginosa EMS1. The new strain produced surface-active rhamnolipids by batch cultivation in mineral salts medium with waste flying oils. The mutant strain KH7, designated P. aeruginosa EMS1, derived by random mutagenesis with N-methyl-N-nitro-N-nitrosogoanidine treatment producing high levels of the biosurfactants was selected by an ion-pair plate assay. The mutant strain KH7 showed 4-5 times more hydrocarbon emulsification as compared to the parent when grown on waste frying oils and various hydrocarbons. Furthermore, P. aeruginosa EMS1 and mutant strain KH7 was also able to use whey as a co-substrate for growth and biosurfactant production. As results of this study, mutant strain KH7 is a very efficient biosurfactant producer, and its culture conditions are relatively inexpensive and economical. Rhamnolipid is synthesized by the rhlAB-encoded rhamnosyltransferase. To be convinced of these genes, we performed PCR based on P. aeruginosa PAO1 whole-genome database. rhl gene cluster nucleotide and amino acid sequences were compared for both parent and mutant. Comparison of nucleotide sequence of rhlAB, there were usually terminal's codons exchange.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.94.2-95
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• 2003

E. intermedium 60-2G possessing a strong ability to solubilize insoluble phosphate, has plant growth-promoting activity, induced systemic resistance activity against scab pathogen in cucumber, and antifungal activity against various phytopathogenic fungi. The phosphate solubilizing activity of 60-2G may be mainly accomplished by production of gluconic acid through a direct extracellular oxidation of glucose by glucose dehydrogenase that required a PQQ cofactor for its activation. A pqq gene cluster conferred Phosphate-solubilizing activity in E. coli DH5${\alpha}$ was cloned and sequenced. The 6,783 bP pqq sequence had six open reading frames (from A to F) and showed 50-95％ homology to pqq genes from other bacteria. The E. coli strain expressing the pqq genes solubilized phosphate from hydroxyapatite after a pH drop to 4.0, which paralleled in time the secretion of gluconic acid. To study the role of PQQ in biocontrol traits of E. intermedium, PQQ mutants of 60-2G were constructed by marker exchangee mutagenesis. The PQQ mutants of E. intermedium were lost activities of solubilizing phosphate, growth inhibition of phytopathogenic fungi, and plant growth promotion. These findings suggest that PQQ plays an important role, possibly activation of certain enzymes, in several beneficial bacterial traits of E. intermedium by as yet an unknown mechanism.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• BMB Reports
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• v.35 no.5
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• pp.498-507
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• 2002

A gene that encodes a thermostable protease, coined thermicin, has been isolated from Thermoanaerobacter yonseiensis that is expressed and characterized in E. coli.. In order to elucidate the molecular characteristics on thermostability of the enzyme, molecular modeling and mutagenesis technology were applied. In the modeling structure, the structural core, including the active site, was well conserved; whereas, the two loop regions were unique when compared to thermitase. The mutant enzyme with the small loop deleted (D190-I196), based on modeling structural information, showed identical enzyme activity. However, when the large loop was deleted (P233-P244), a little lower $K_m$ and even a lower kcat was found. This indicates that the large loop could influence catalytic activity. However, the unfolding temperature ($T_m$), which was determined by a differential-scanning calorimetry for the mutant enzyme deleted the small loop, was $96^{\circ}C$. This is $14^{\circ}C$ lower than that for the parent thermicin. These results suggest that the small loop may play a role in maintaining the proper folding of the enzyme at high temperatures, whereas the large loop might be related to catalysis.

• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.277-283
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• 2015

Plant genome analyses, including Arabidopsis thaliana showed a large gene family of plant receptor kinases with various extracellular ligand-binding domain. Now intensively studies to understand physiological and cellular functions for higher plant receptor kinases in diverse and complex biological processes including plant growth, development, ligands perception including steroid hormone and plant-microbe interactions. Brassinosteroids (BRs) as a one of well know steroid hormone are plant growth hormones that control biomass accumulation and also tolerance to many biotic and abiotic stress conditions and hence are of relevance to agriculture. BRI1 receptor kinase, which is localized in plasma membrane in the cell sense BRs and it bind to a receptor protein known as BRASSINOSTEROID INSENSITIVE 1 (BRI1). Recently, we reported that BRI1 and its co-receptor, BRI1-ASSOCIATED KINASE (BAK1) autophosphorylated on tyrosine residue (s) in vitro and in vivo and thus are dual-specificity kinases. Other plant receptor kinases are also phosphorylated on tyrosine residue (s). Post-translational modifications (PTMs) can be studied by altering the residue modified by directed mutagenesis to mimic the modified state or to prevent the modification. These approaches are useful to not only characterize the regulatory role of a given modification, but may also provide opportunities for plant improvement.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.144-150
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• 1997

After DNA damage, umuDC is the only SOS operon that must be induced to promote SOS mutagenesis in Escherichia coli. The recombinant plasmid pBC401 and pBC402 were constructed to fuse the lac structural genes with promoter region of umuDC operon to induce the expression of lacZ gene by DNA damage. We transformed the plasmid pBC401 and pBC402 into E. coli MC1061, lacZ deleted strain and determined the activity of $\beta$-galactosidase for various mutagen; UV, mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroqunoline-1-oxide (NQO), ethyl methanesulfonate (EMS). The $\beta$-galactosidase activities of PBC401 and pBC402 for UV, MMC, and NQO were increased in proportion to expression time until 3 hours thereafter, the activities were constant or slightly decreased. The activities for MNNG and EMS were not so high as for UV, MMC, and NQO. When MNNG and EMS were treated, $\beta$-galactosidase activity of pBC402 was slightly lower than pBC401 but when UV, MMC, and NQO were treated in pBC402, $\beta$-galactosidase activity was slightly higher than in pBC401. Therefore, the pBC402 was better than the pBC401 in terms of sensitivity for frameshift mutagen. We suggest that the plasmid pBC401 and pBC402 are easy to detect mutagens which cause frameshift mutation rather than point mutation.

• BMB Reports
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• v.45 no.12
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• pp.748-753
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• 2012

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3'-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus ($TMGS^+$). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and $TMGS^+$, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1563-1567
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• 2007

AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (${\Delta}afsK$-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.