• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.1-8
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• 2018

Gul (oyster) jeotgals (GJs) were prepared using different types of salt (23%, w/v): purified salt, solar salt aged for 3 years, and bamboo salt crystalized 3 times. One set of GJs was fermented with Bacillus subtilis JS2 ($10^6CFU/g$), while the other GJ set was fermented without starter. During fermentation for 24 weeks at $15^{\circ}C$, the starter GJs showed 10-fold higher bacilli counts than the no-starter GJs, where the maximum bacilli count was $8{\times}10^3CFU/g$. All 28 bacilli strains isolated from the 6-week GJs were identified as B. subtilis by using a RAPD-PCR, indicating that some of the B. subtilis JS2 cells remained viable. Lactic acid bacteria (LAB) and yeasts were present at low levels, $10^1-10^2CFU/g$. LAB with protease activities isolated from 10-week samples were identified as Enterococcus species. The isolates obtained at 16 weeks were all Staphylococcus species. The GJs with bamboo salt showed higher pH and lower titratable acidity (TA) values than the other GJs due to the strong alkalinity of bamboo salt. The amino-type nitrogen in the GJs increased slowly during the fermentation. At 24 weeks, the GJs with purified salt showed the highest amino-type nitrogen (412-430 mg%), followed by the GJs with solar salt (397-406 mg%) and GJs with bamboo salt (264-276 mg%). Meanwhile, the GJs with bamboo salt showed the highest ammonia-type N (63.67 mg%), followed by the GJs with purified salt (49 mg%) and solar salt (48 mg%).

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.273-282
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• 2000

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-l-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of ${\Delta}rmlC$ E. coli strain $S{\Phi}874$ (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

• Journal of Life Science
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• v.19 no.9
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• pp.1218-1225
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• 2009

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at $55^{\circ}C$. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less $\alpha$-helix and more $\beta$ strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1482-1492
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• 2018

Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino acid sequences added to the N-terminus of FOT and expressed in P. pastoris. The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature ($40^{\circ}C$) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at $40^{\circ}C$ and $50^{\circ}C$. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation ${\pi}$-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, $k_{cat}/K_m$), 1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198, and G208) and catalytic triad (His42, Asp102, and Ser180), which would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.

• Bulletin of the Korean Chemical Society
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• v.12 no.3
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• pp.332-334
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• 1991

The reaction and coordination chemistry of chiral ferrocenylaminophosphines such as 2-(diphenylphosphino)-1-(N,N-dimethylaminoethyl) ferrocene (PPFA), and 1',2-bis(diphenylphosphino)-l-(N,N-dimethylaminoe thyl) ferrocene (BPPFA), with various iron carbonyls have been investigated. PPFA reacted with iron carbonyls, Fe$(CO)_5$, $Fe_2(CO)_9$, or $Fe_3(CO)_{12}$ to give an iron complex of the type (${\eta}^1$-PPFA-P)Fe$(CO)_4$ (1) as a single product regardless the choice of the iron carbonyls. The bisphosphine ligand BPPFA afforded two products (${\eta}^1$-BPPFA-P)Fe$(CO)_4$ (2) and (${\mu}$,${\eta}^1$-BPPFA-P,P)$Fe_2(CO)_8$ (3) in which BPPFA acts as a monodentate and a bridging ligand, respectively. In all cases coordination to the -Fe$(CO)_4$ moiety is made through the phosphine rather than the amino group and, in the case of 2, the coordination is made through the phosphine substituted at the $C_5H_4$ ring to reduce the steric congestion.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.9.1-9.7
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• 2012

Filamentous phages have been in the limelight as a new type of nanomaterial. In this study, genetically and chemically modified fd phage was used to generate a biomimetic phage self-assembly product. Positively charged fd phage (p8-SSG) was engineered by conjugating 3-aminopropyltriethoxysilane (APTES) to hydroxyl groups of two serine amino acid residues introduced at the N-terminus of major coat protein, p8. In particular, formation of a phage network was controlled by changing mixed ratios between wild type fd phage and APTES conjugated fd-SSG phage. Assembled phages showed unique bundle and network like structures. The bacteriophage based self-assembly approach illustrated in this study might contribute to the design of three dimensional microporous structures. In this work, we demonstrated that the positively charged APTES conjugated fd-SSG phages can assemble into microstructures when they are exposed to negatively charged wild-type fd phages through electrostatic interaction. In summary, since we can control the phage self-assembly process in order to obtain bundle or network like structures and since they can be functionalized by means of chemical or genetic modifications, bacteriophages are good candidates for use as bio-compatible scaffolds. Such new type of phage-based artificial 3D architectures can be applied in tuning of cellular structures and functions for tissue engineering studies.

• Journal of Microbiology
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• v.35 no.3
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• pp.206-212
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• 1997

Heliobacillus mobilis is a strictly anaerobic Gram-positive bacterium which contains a primitive Photosystem I-type reaction center. The membrane-bound cytochrome $C_{553}$ from the heliobacterium suggested to be the immediate electron donor to the photooxidized pigment (P798+) has been isolated and characterized. The heme protein was visualized as a major component with an apparent molecular size of 17kDa in TMBZ-staining analysis of the membrane preparation and showed characteristic $\alpha$ (552.5 nm), $\beta$ (522nm), and Soret absorption (416 nm) peaks of a typical reduced c-type cytochrome in the partially purified sample. The internal 43 amino acid sequence of the electron donor was obtained by chemical agent and protease treatments followed by N-terminal sequencing of the resulting fragments. The internal sequence carries lots of lysine residues and a Cys-X-X-Cys-His sequence motif which are the characteristics of typical c-type cytochromes. The analysis of the sequence by FAST or FASTA program, however, did not show any significant similarity to other known heme proteins.

• Molecules and Cells
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• v.21 no.1
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• pp.21-28
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• 2006

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase PKR plays a key role in interferon-mediated host defense against viral infection, and is implicated in cellular transformation and apoptosis. We have isolated a cDNA of simian PKR encoding a product with 83% amino acid identity to the human homolog and showed that PKR expression is significantly attenuated in the Vero E6 African green monkey kidney cells devoid of type I interferon genes. A variant form of PKR lacking the exon 12 in the kinase domain is produced in these cells, presumably from an alternatively spliced transcript. Unlike wild type PKR, the variant protein named PKR-${\Delta}E12$ is incapable of auto-phosphorylation and phosphorylation of eIF2-${\alpha}$, indicating that the kinase sub-domains III and IV embedded in exon 12 are indispensable for catalytic function. PKR-${\Delta}E12$ had no dominant negative effect but was weakly phosphorylated in trans by wild type PKR.

• BMB Reports
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• v.33 no.3
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• pp.249-255
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• 2000

The Haemophilus influenzae type b (Hib) has been a major cause of bacterial meningitis in children who are less than two years old. The variable (V) region repertoire of adult Caucasian antibodies (Abs) to Hib polysaccharide (PS) has been characterized well. The majority of adult antibodies against Hib uses VL that is derived from the Vk gene A2 and have arginine at the N region. In order to explore the possibility those antibody responses to Hib-PS is variable in various age groups, we examined the VL regions of the antibodies to Hib-PS in Korean adults and children. We immunized Korean adults (n = 8) and children (n = 39) with Hib tetanus conjugated vaccines, isolated RNAs from the peripheral lymphocytes, and amplified the A2-derived VL regions by RT-PCR. The PCR products were subcloned and sequenced. Forty-seven out of 54 independent clones from children used the $J{\kappa}2$, or $J{\kappa}3$ gene in preference. The adults, however, used all of the $J{\kappa}$ genes evenly. With respect to the amino acid sequences of variable regions, adult $A2-J{\kappa}$ recombinants have a germline sequence. But, the 76th codon (AGC) of child $A2-J{\kappa}2$ recombinants was substituted with CGC (arginine) in most cases (88 %) and 77 percent of child clones using the $A2-J{\kappa}3$ genes have isoleucine-109 at the junction of $J{\kappa}-C{\kappa}$ instead of threonine that is found in a germline sequence. These results suggest that the mechanism of antibody production in young children is different from that of adults.

• BMB Reports
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• v.41 no.7
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• pp.516-522
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• 2008

The glycation of BSA leads to protein/peptide modifications that result in the formation of AGEs. AGEs react with the amino groups of N-terminal amino acid residues, particularly arginine and lysine residues. Enhanced AGE formation exists in the blood and tissues of diabetics, as well as in aging and other disorders. The Identification of AGEs is of great importance. Mass spectrometry has been applied to identify and structurally elucidate AGEs. Here, we report on the identification of AGE-peptides and AGE precursors based on relative mass changes as a result of specific AGE formation. HPLC-ESIMS, ESI-MS/MS, and the Mascot database were used. The relative mass changes due to the specific type of AGE formation were added to the identified peptides followed by a manual search of the glycated samples, which resulted in the identification of seven peptides for the formation of five AGEs, namely CML, pyrraline, imidazolone A, imidazolone B, and AFGP. Four glycated peptides (FPK, ECCDKPLLEK, IETMR, and HLVDEPQNLIK) were identified in the formation of AGE-precursors.