• Journal of Life Science
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• v.22 no.1
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• pp.7-15
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• 2012

A polyguluronate-specific lyase from Streptomyces sp. strain M3 has been previously cloned and characterized. In this study, the M3 alginate lyase gene in the pColdI vector was mutated by site-directed mutagenesis and random mutagenesis to enhance the alginate degrading activity. Six mutants were obtained: Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu, and Gly194Cys. Phe99Leu and Lys191Glu mutants completely lost their alginate lyase activity, whereas the alginate degrading activity of Gly194Cys mutant increased by nearly 10 fold. The 3-D protein structure of M3 alginate lyase, which was constructed using the Swiss-Model automodeler, was also compared to the crystal structure of another alginate lyase. A mutated glycine residue was positioned between Gly193 and Tyr195 of the C-terminal conserved sequence, YFKAGXYXQ. A phenylalanine residue (at position 99) and a glycine residue (at position 194) mutated in this study were distant from the active site, but the degrading activity was strongly affected by their mutation.

• Bulletin of the Korean Chemical Society
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• v.30 no.6
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• Bulletin of the Korean Chemical Society
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• v.30 no.7
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• pp.1547-1552
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• 2009

Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.305-311
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• 1988

The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 $spr^{r}$ chromosome at the frequency of $1.0\times 10^{-5}-5.0\times 10^{-7}$ and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin ($50{\mu}$g/ml) and spectinomycin ($100{\mu}$g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1077-1086
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• 2016

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn²⁺ enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp¹⁷¹, His²⁵⁴, and His²⁷¹) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60℃ and pH 8.0 and towards DHA at 60℃ and pH 6.0. DHA reduction was the dominant reaction, with a lower K_m(DHA) of 1.08 ± 0.13 mM and V_max of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a K_m(glycerol) of 30.29 ± 3.42 mM and V_max of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60℃. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg⁴³, both related to dinucleotide binding.

• 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.18 no.4
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• pp.631-638
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• 2008

Tandem affinity purification (TAP) method combined with LC-MS/MS is the most accurate and reliable way to study the interaction of proteins or proteomics in a genome-wide scale. For the first time, we used a TAP-tag as a mutagenic tool to disrupt protein interactions at the specific site. Although lots of commonly used mutational tools exist to study functions of a gene, such as deletional mutations and site-directed mutagenesis, each method has its own demerit. To test the usefulness of a TAP-tag as a mutagenic tool, we applied a TAP-tag to RNA polymerase II, which is the key enzyme of gene expression and is controlled by hundreds of transcription factors even to transcribe a gene. Our experiment is based on the hypothesis that there will be interrupted interactions between Pol II and transcription factors owing to the TAP-tag attached at the C-terminus of each subunit of Pol II, and the abnormality caused by interrupted protein interactions can be observed by measuring a cell-cycle of each yeast strain. From ten different TAP-tagged strains, Rpb7- and Rpb12-TAP-tagged strains show severe defects in growth rate and morphology. Without a heterodimer of Rpb4/Rpb7, only the ten subunits Pol II can conduct transcription normally, and there is no previously known function of Rpb7. The observed defect of the Rpb7-TAP-tagged strain shows that Rpb7 forms a complex with other proteins or compounds and the interruption of the interaction can interfere with the normal cell cycle and morphology of the cell and nucleus. This is a novel attempt to use a TAP-tag as a proteomic tool to study protein interactions.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.201-211
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• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.505-512
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• 2002

대장균 K99 섬모의 생합성은 8개로 구성된 K99의 특이 유전자의 발현과 숙주유래 인자에 의해 조절되는 다른 유전자들의 발현에 의존된다. 본 연구에서는 K99섬모 유전자군중 제 3지역 발현에 유전조절자의 관련성 여부를 연구하였다. Gel retardation 분석 방법올 통하여 제3지역의 발현에 관련된 유전조절단위를 함유한 fanF 지역의 단백질 인자가 부착됨을 암시하였다. 이 분석방법을 이용한 결과는 또한 이 단백질 인자가 K99 유전자에서 유래되지 않고 대장균 염색체에서 유래됨을 지적하였다. 이를 보다 더 조사하기 위하여 대장균 염색체에 Tn10 transposon 유전자 변이 실험을 수행하였다. K99 유전자군으로부터 제 1지역과 제2지역의 유전자를 제거시키고, 제 3지역의 유전자인 fanG에 transposon TnlacZ를 삽입한 pTL65-1 plasmid을 제작하였다. 이 pTL65-1는 다시 Tn10으로 염색체가 변이된 대장균에 주입하였다. 3개의 pTL65-1 주입된 Tn10 대장균 변이체 내에서 fanG의 발현이 증가되었다. 이들 변이대장균으로부터 Tn10이 어떤 염색체 유전자 부위를 변이 시켰는지 확인하기 위해서 변이부위 유전자를 cloning하여 염기서열을 분석하였다. 이중 2개의 clone이 동일하였으며 지금까지 알려지지 않은 유전자였다. 이들 2개의 변이체 내에서 fanG의 발현은 대조군과 비교해 약 4.2배 증가 되였다. 결론적으로 이들 2개의 clone으로부터 유래된 인자는 지금까지 알려지지 않은 제 3지역의 억제 조절자임을 나타내었다.