• 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 Life Science
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• v.14 no.4
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• pp.636-643
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• 2004

D-Xylose isomerase produced by Lactococcus sp. JK-8, isolated from kimchi, was purified 17-fold of homogeneity, and its physicochemical properties were determined. Although the N-terminal amino acid sequence of D-xylose isomerase was analysed to Ala-Tyr-Phe-Asn-Asp-Ile-Ala-Pro-Ile-Lys, it was not similar to that of Lactobacillus enzyme. The molecular weight of the purified enzyme was estimated to be 180 kDa by gel filtration, 45 kDa by SDS-PAGE and the enzyme was homotetramer. The optimum pH of the enzyme was around 7 and stable between pH 6 and 8. The optimum reaction temperature was 7$0^{\circ}C$ and stable up to 7$0^{\circ}C$ in the presence of 1 mM $Mn^{2+}$. Like other D-xylose isomerases, this enzyme required divalent cation, such as $Mg^{2+}$, $Co^{2+}$, or $Mn^{2+}$ for the activity and thermostability. $Mn^{2+}$was the best activator. Substrate specificity studies showed that this enzyme was highly active on D-xylose. The enzyme had an isoelectric point of 4.8, and fm values for D-xylose was 5.9 mM.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.220-228
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• 1995

For polyhydroxyalkanoic acid (PHA) production, several microorganisms were isolated from sewage sludge. One of them, GB-77 strain, was chosen from its PHB/HV copolymer production on only fructose without cosubstrate. The isolated strain GB-77 was identified as the genus Alcaligenes. Optimal temperature and pH for cell growth were 36C and 6.8. Optimal medium composition was 10 g/l of fructose and 5 g/l of polypeptone, 1 $\times$ 10$^{-2}$M Na$^{2}$HP0$^{4}$, 1.3 $\times$ 10$^{-2}$M KH$^{2}$PO$^{4}$. To investigate the optimal condition for polyhydroxyalkanoic acid production two-stage culture technique was used; first stage for cell growth and second stage for PHA production on unbalanced growth conditions. Optimal conditions for high PHA production were C/N ratio 50, temperature 36$\circ$C and pH 6.8. To overcome fructose inhibition on cell growth, intermittent feeding fed-batch culture technique was used. Total cell concentration was 17.4 g/l with 9.1 g/l of PHA. The purified PHA was identified PHB/HV copolymer by NMR analysis.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.452-458
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• 1996

A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.264-271
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• 2006

Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature ($40^{\circ}C$), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below $30^{\circ}C$, whereas FFP2 was very stable in the pH range of 4-11 and temperature below $40^{\circ}C$. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of $CO^{2+}$ and $Zn^{2+}$ and inhibited by $Cu^{2+},\;Ni^{2+}$, and $Hg^{2+}$. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the $A{\alpha}$- and $B{\beta}$-chains of fibrinogen over ${\gamma}$-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not. $K_{m}$ and $V_{max}$ values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.350-355
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• 2010

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.50-58
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• 1996

Interspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several autotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were $3{\times}10^{-5}{\sim}1{\times}10^{-5}$. From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80$^{\circ}C$. The enzymatic activity was increased by the presence of $Mg^{2+}$ and $Mn^2$ ions.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.9
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• pp.1248-1252
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• 2012

Potassium added with bamboo salt showed better antioxidative effects than bamboo salt, solar salt, or purified salt. It also showed inhibitory effects on the mutagenicity of MNNG (N-methyl-N-nitro-N-nitrosoguanidine) in a Salmonella Typhimurium TA100 tester strain. At concentrations of 1.25 and 2.5 mg/plate, potassium bamboo salt and bamboo salt showed weaker co-mutagenicity effects than either purified salt or solar salt, respectively. Anticancer effects of salts were evaluated using MTT assay in HCT-116 human colon carcinoma cells. At a 1% salt concentration, the growth inhibitory rate of potassium bamboo salt was 54%, higher than that of 1 time baked bamboo salt (36%). However, purified salt and solar salt showed relatively lower inhibitory effects of 19% and 23%, respectively. To determine the inhibitory mechanisms of potassium bamboo salt, the expression levels of Bax and Bcl-2 genes in HCT-116 cells were determined by RT-PCR. Potassium bamboo salt significantly increased Bax and decreased Bcl-2 expression levels unlike bamboo salt, purified salt, and solar salt (p<0.05). Therefore, addition of potassium to salt decreased co-mutagenicity and increased in vitro antioxidative and anticancer effects.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.35-45
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• 1989

Streptococcus thermophilus 510 was investigated as n potential source of $\beta$-galactosidase. Optimum cultural conditions for maximum enzyme production were 0.5% loctose as carbon source, initial pH 7.0, 37 $^{\circ}C$, and 18 hours of cultivation. The enzyme was purified to homogeneity by ammonium sulfate fractionation, protamine sulfate precipitation, Sephadex G-200 gel filtration, and DEAE-Sephadex A-50 ion exchange chromntography. The purified enzyme exhibited an optimum pH at 1.0, and an optimum temperature of 5$0^{\circ}C$. Metal ions such as Mn$^{2+}$ and $K^+$, dithiothreitol, and 2-mercaptoethanol stimulated $\beta$-galactosidase activity. Ethylenediamine tetraacetic add, 8-hydroxyquinoline, Hg$^2+$, Zn$^{2+}$, Co$^{2+}$, $Ca^{2+}$, and galactose were inhibitory. The $K_m$ and V$_{max}$ for o-nitrophenyl $\beta$-D-galactopyranoside were 1.25mM and 88.50$\mu$moles/min.mg protein, respectively. The molecular weight was estimated to be 520,000, and the amino acid composition indicated relatively high contents of glutamic acid, aspartic acid, leucine, and valine.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.51 no.5
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• pp.408-419
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• 2006

A pot experiment was conducted for two years to evaluate the effects of purified Si fertilization combined with $^{15}N$ on the nutrient uptake, plant growth characteristics, and photosynthetic characteristics of rice in water melon cultivated soil. In 2002, plant height was positively affected at 25 DAT (Day After Transplanting) by Si fertilization in 100%N treatment. However, in 2003, plant height at 25 DAT was negatively affected by Si fertilization in low N level but it was reversed in high N level with initial increase of plant height. Tiller number per pot was positively affected by N and Si fertilization level, especially for high N fertilized treatment. Leaf color was positively affected by Si fertilizatlon in no N fertilized pots, however, Si was not effected in 50%N and 100%N fertilized treatments. N harvest index (NHI) increased with increased Si fertilization in no N plots, however it decreased with increasing of N fertilization level. Nitrogen use efficiency (NUE) decreased with increasing of fertilized N but Si fertilization increased NUE in 50%N plots, however, it was not different by the Si fertilization level in 100%N plots. In 50%N+200%Si plots, NUE was greatest with 130 and shoot N content was $16.2g-N/m^{2}$. N content ($g/m^{2}$) in rice plant increased with increasing Si fertilization in no N plots at panicle initiation stage, 50 and 100%N plots at heading stage and all N treatment at harvesting time. This was mostly more efficient in late growth stage than early growth stage. The concentration (%) of P and K increased with increasing N fertilization level at heading and harvesting but it was not significantly different by the Si fertilization treatment except a little decreasing with increasing Si fertilization level at heading. Potassium content was also not significantly related with N fertilization level except increasing with Si fertilization level at panicle initiation stage. Plant Ca content (%) decreased with increasing of Si fertilization at heading stage and Si fertilization increased Ca content at panicle initiation stage and heading stage and it increased with increasing of Si fertilization level. Photosynthetic activity was not directly related with Si fertilization amount, however, Fluorescent factors, Fv'/Fm' and PsII, were positively affected by Si fertilization level. In conclusion, N fertilization in Si 200% fertilized condition should be reduced by about 50% level of recommended N fertilization for rice cropping in green-house water-melon cultivated paddy field. However, improvement of Ps by Si fertilization could not be attributed to Ps activity in the same leaf area but because of increased total leaf area per pot improved fluorescent characteristics.