• 제목/요약/키워드: protein cyclization

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Protein Cyclization Enhanced Thermostability and Exopeptidase-Resistance of Green Fluorescent Protein

  • Zhao, Zhonglin;Ma, Xin;Li, Liang;Zhang, Wei;Ping, Shuzhen;Xu, Ming-Qun;Lin, Min
    • Journal of Microbiology and Biotechnology
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    • 제20권3호
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    • pp.460-466
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    • 2010
  • A mutant of green fluorescent protein ($GFPmut3^*$) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of $GFPmut3^*$ was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear $GFPmut3^*$. The circular $GFPmut3^*$ was $5^{\circ}C$ more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular $GFPmut3^*$ also displayed increased relative fluorescence intensity. In addition, chemical stability of $GFPmut3^*$ against GdnHCl revealed more stability of the circular form compared with the linear form.

Analysis of the functional domains of CFTase gene cloned from Xanthomonas oryzae #5 using recombinant deletion mutant

  • Kim, Byeong-U;Yu, Dong-Ju;Ryu, Hye-Gyeong;Park, Ju-Hui
    • 한국생물공학회:학술대회논문집
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    • 한국생물공학회 2001년도 추계학술발표대회
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    • pp.727-730
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    • 2001
  • Various recombinant deletion mutants were constructed from cycloinulo - oligosaccharide fructanotransferase(CFTase) gene of Xanthomonas oryzae #5 . The mutants were expressed in Escherichia coli DH5${\alpha}$. We were able to obtain three recombinant proteins were purified, and examine their CFTase and hydrolyzing activity. N-terminal deletion mutant had both CFTase activity and hydrolyzing activity. however, in C-terminal and N,C-terminal deletion mutant disappeared CFTase activity, but hydrolyzing activity remained. From there results, it seems that the C-terminal region(amino acid $1173{\sim}1333$) is important for cyclization.

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Mutant cAMP Receptor Protein Binds to DNA without DNA Bending (DNA 벤딩(휨) 없이 돌연변이 cAMP 수용체 단백질의 결합)

  • Gang, Jong-Back
    • Journal of Life Science
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    • 제16권7호
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    • pp.1225-1228
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    • 2006
  • Cyclic AMP receptor protein (CRP) complexed with cAMP binds to DNA and induces sharp DNA bending around ${\sim}90$ degree. Previous publication (5), however, reported that mutant CRP:cGMP complex showed high migration rate relative to mutant CRP:cAMP complex on native polyacrylamide gel. To confirm DNA structural change in the presence of CRP and cyclic nucleotide, molar cyclization factor $(j_M)$ [13] was measured with 6 constructed DNA fragments. Nonlinear regression analysis of $j_M$ data indicated that mutant CRP did not induce DNA bending in the presence of cGMP but bent DNA in the presence of cAMP without any helical twist change in DNA.

Expression of Bacillus macerans Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae.

  • Kim, Gyu-Yong;Kim, Myeong-Dong;Han, Nam-Su;Seo, Jin-Ho
    • 한국생물공학회:학술대회논문집
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    • 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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    • pp.191-193
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    • 2000
  • A whole-cell biocatalyst was constructed by immobilizing an enzyme on the surface of the yeast Saccharomyces cerevisiae. The gene encoding Bacillus macerans cyclodextrin glucanotransferase(CGTase) was fused with the AGA2 gene encoding a small peptide disulfide-linked to the aga1, a cell wall protein of a-agglutinin. The plasmid was introduced S. cerevisiae and expressed in the medium consisting of 10g/L yeast extract, 20g/L peptone, and 20g/L galactose. The activity was detected with the formation of cyclodextrin(CD) from 10g/L soluble starch. Surface display of CGTase was also verified with the halo-test, flow cytometry, and immunofluorescence microscopy. The recombinant S. cerevisiae produced ${\alpha}-cyclodextrin$ more efficiently than the free CGTase by simultaneous fermentation and cyclization as yeast consumes glucose and maltose which are inhibitors for CD synthesis.

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Comparison of Catalyzing Properties of Bacterial 4-α-Glucanotransferases Focusing on Their Cyclizing Activity

  • Kim, Jung-Eun;Tran, Phuong Lan;Ko, Jae-Min;Kim, Sa-Rang;Kim, Jae-Han;Park, Jong-Tae
    • Journal of Microbiology and Biotechnology
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    • 제31권1호
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    • pp.43-50
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    • 2021
  • A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

Enzymatic Properties of Cyclodextrin Glycosyltransferase from Alkalophilic Bacillus sp. YC-335 (호알칼리성 Bacillus sp.가 생산하는 Cyclodextrin Glycosyltransferase의 효소적 특성)

  • Jung, Yong-Joon;Jung, Myeong-Ho;Yu, Ju-Hyun
    • Korean Journal of Food Science and Technology
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    • 제23권1호
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    • pp.93-97
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    • 1991
  • The enzymatic properties of purified CGTase from alkalophilic Bacillus sp. YC-335 have been examined. Apparent Vmax values of the enzyme in transferring glycosyl residues ${\alpha}-,\;{\beta}-and\;{\gamma}-cyclodextrin(CD)$ to sucrose were $16.13,\;21.8\;and\;9.8{\mu}moles/min/mg\;protein$, respectively and Km values of the corresponding CD were 1.68, 0.33 and 0.37 mM, respectively. A number of saccharides, specially starch hydrolyzates such as glucose and maltose, could activate the dextrinizing activity of the enzym. However, the dextrinizing activity was inhibited by ${\beta}-CD$. It was found from Lineweaver-Burk plot that the inhibition of CGTase by ${\beta}-CD$ was noncompetitive. High performance liquid chromatographic analysis showed that the enzyme has three kinds of activity ; transglycosylation and disproportionation as well as cyclization.

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The Facile and Efficient Synthesis of 8-Chloroadenosine $3^I,5^I$-cyclic monophosphate by phosphorylative cyclization of 8-chloroadenosine and its characterization by$^1 H and 13^C$ NMR spectroscopy

  • Woo, Nam-Tae;Jin, Sun-Yong;Cho, Dae-Jin;Kim, Nam-Sun;Bae, Eun-Hyung;Jung, Jee-Hyung;Ham, Won-Hun;Jung, Young-Hoon
    • Archives of Pharmacal Research
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    • 제20권2호
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    • pp.176-179
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    • 1997
  • Purine nucleosides were chlorinated by the reaction of acyl chloride in DMF with MCPBA under mild conditions with moderate yields. And, satisfactory method for the synthesis of ribonucleoside-$3^{I},5^{I}$-cyclic phosphates and its characterization by$^{1}H$ and $^{13}C$ nmr spectroscopy is described.

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Cloning and Characterization of Cycloinulooligosaccharide Fructanotransferase (CFTase) from Bacillus polymyxa MGL21

  • Jeon, Sung-Jong;You, Dong-Ju;Kwon, Hyun-Ju;Shigenori Kanaya;Namio Kunihiro;Kim, Kwang-Hyeon;Kim, Young-Hee;Kim, Byung-Woo
    • Journal of Microbiology and Biotechnology
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    • 제12권6호
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    • pp.921-928
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    • 2002
  • Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.

Identification of Korean Ginseng Cytochrome P450 gene and Its Characterization by Transformation System (고려인삼 유래 Cytochrome P450 유전자의 동정 및 형질전환에 의한 특성검정)

  • Shim, Ju-Sun;Kim, Yu-Jin;Jung, Seok-Kyu;Kwon, Woo-Saeng;Kim, Se-Young;Yang, Deok-Chun
    • Journal of Ginseng Research
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    • 제33권3호
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    • pp.212-218
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    • 2009
  • Triterpenoid saponins were synthesized in Panax ginseng C.A. Meyer via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoids are backbone and undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450 (CYP)-dependent monooxygenases, glycosyltransferase and other enzymes. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. A cDNA clone encoding a putative CYP gene was isolated from flower bud of ginseng and transformed into the plant(Nicotiana tabacum cv. Xanthi) and confirmed by PCR analysis. The CYP gene (PgCYP) contained an open reading frame(ORF) encoding mature protein of 500 amino acids. The expression of PgCYP were investigated in transgenic tobacco by reverse transcriptase-polymerase chain reaction (RT-PCR).

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2016년도 춘계학술대회 및 임시총회
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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