• Applied Biological Chemistry
• /
• v.27 no.4
• /
• pp.271-277
• /
• 1984

Molecular weight of Exo-maltotetraohydrolase produced by Pseudomonas stutzeri IAM 12097 was estimated to be approrimately 63,000 and 60,000 with SDS-polyacrylamide gel electrophoresis and Sephadex-G-100 gel filtration, respectively. The isoelectric point was appeared to be pH 4.8. Optimum pH, the stable pH range and optimum temperature of this enzyme were pH 6.6, $pH6.0{\sim}10.5\;and\;45{\sim}50^{\circ}C$. The enzyme was stable below $40^{\circ}C$ and was rapidly inactivated above $55^{\circ}C$. This enzyme was inactivated completely by $Ag^+,\; Hg^{++},\;I_2$ and ${\beta}-cycoldextrin$, and slightly by EDTA, ${\rho}-CMB$ and IAA. Michaelis constant(Km) of this enzyme toward soluble starch, amylose and amylopectin were 7.70mg/ml, 6.17mg/ml, 5.56mg/ml, respectively.

• BMB Reports
• /
• v.29 no.5
• /
• pp.455-461
• /
• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.

• BMB Reports
• /
• v.30 no.1
• /
• pp.46-54
• /
• 1997

Alkaliphilic Bacillus sp. S-1 secretes a large amount (approximately 80% of total pullulanase activity) of an extracellular pullulanase (PUL-E). The pullulanase exists in two forms: a precursor form (PUL-I: $M_r$ 180,000), and a processed form (PUL-E: $M_r$ 140,000). Two forms were purified to homogeneity and their properties were compared. PUL-I was different in molecular weight, isoelectric point, $NH_2$-terminal amino acid sequence, and stabilities over pH and temperature ranges. The catalytic activities of PUL-I were also distinguishable in the $K_m$ and $V_{max}$ values for various substrates, and in the specific activity for pullulan hydrolysis. PUL-E showed 10-fold higher specific activities than PUL-I. However. PUL-I is immunologically identical to PUL-E, suggesting that PUL-I is initially synthesized and proteolytically processed to the mature form of PUL-E. Processing was inhibited by PMSF, but not by pepstatin, suggesting that some intracellular serine proteases could be responsible for processing of the PUL-I. PUL-I has a different conformational structure for antibody recognition from that of PUL-E. It is also postulated that the translocation of alkaline pullulanase(AP) in the bacterium possibly requires processing of the $NH_2$-terminal region of the AP protein. Processing of the precursor involves a conformational shift. resulting in a mature form. Therefore. precursor processing not only cleaves the signal peptide, but also induces conformational shift. allowing development of active form of the enzyme.

• BMB Reports
• /
• v.37 no.6
• /
• pp.684-690
• /
• 2004

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and $50^{\circ}C$. It was rather stable within the range of 25 to $50^{\circ}C$ but lost activity rapidly above $50^{\circ}C$. The enzyme was activated by $Co^{2+}$ or EDTA and stabilized by dithiothreitol. The kinetic constants, $K_m$ and $V_{max}$ for heparin were $1.3{\times}10^{-5}\;M$ and $8.8\;{\mu}mol/min{\cdot}mg$. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

• Applied Chemistry for Engineering
• /
• v.8 no.5
• /
• pp.830-836
• /
• 1997

The surface modification of iron oxide particle produced from steel-pickled acid by sodium-contained materials was studied. The molar ratio of $SiO_2$ to $Na_2O$ of sodium silicate was 1, 2, 3.5, respectively. The dispersion stability of iron oxide suspension as functions of amount of silica and pH was evaluated by surface charge and sedimentation velocity of iron oxide particle. Then the amount of sodium silicate was determined to provide a dispersion stability of iron oxide particle above pH 7. Finally, the surface modification of iron oxide particle with sodium silicate as silica-contained materials was done by wet ball milling. In the results of study, the dispersion stability of silica modified iron oxide particle was largely depended on amount of silica and pH together. The untreated iron oxide was unstable at pH 8, i.e. isoelectric point, but, the surface modified iron oxide particle with 0.8wt% silica was stable above pH 5. The dispersion stability was enhanced with 0.2wt% of anionic polyelectrolyte.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.1
• /
• pp.72-79
• /
• 2005

A new Bacillus peptidase, bacillopeptidase DJ-2 (bpDJ-2), with molecular mass of 42 kDa and isoelectric point (pI) of 3.5- 3.7, was purified to homogeneity from Bacillus sp. DJ-2 isolated from Doen-Jang, a traditional Korean soybean fermented food. The enzyme was identified as an extracellular serine fibrinolytic protease. The optimal conditions for the reaction were pH 9.0 and $60^{\circ}C$. The first 18 amino acid residues of the N-terminal amino acid sequence of bpDJ-2 were TDGVEWNVDQIDAPKAW, which is identical to that of bacillopeptidase F (bpf). However, based on their Nterminal amino acid sequence, molecular size, and pI, it is different from that of bpf and extracellular 90 kDa. The whole (2,541 bp, full-bpDJ-2) and mature (1,956 bp, mature-bpDJ-2) genes were cloned, and its nucleotide sequence and deduced amino acid sequence were determined. The expressed proteins, full-bpDJ-2 and mature-bpDJ-2, were detected on SDSPAGE at expected sizes of 92 and 68 kDa, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.31 no.3
• /
• pp.393-398
• /
• 1998

The effect of fermentation period on the accumulation of 55,600 dalton polypeptide was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as a quality parameter of anchovy sauces. Also, proximate compositions, total nitrogen contents, amount of specfic pepited and isoelectric point(pI0 were investigated during fermentation periods. Total nitrogen contents significantly increased until 18 months. Polypeptide of 55,600 and 46,900 dalton on SDS-PAGE and pI 5.2, 5.6 and 6.0 on isoelectric focusing were identified in all the samples. Especially, the amount of 55,600 dalton had no important change during fermentation periods, and it had a high correlation with dilution degree of anchovy sauces diluted with water. The results could be suggest that the amonts of 55,600 dalton polypeptide will be index for quality estimation of commerical anchovy sauces.

• Journal of Plant Biology
• /
• v.39 no.3
• /
• pp.173-177
• /
• 1996

A novel calcium binding protein (CaBP) was purified to electrophoretic homogeneity from Dunaliella salina. In the course of purification experiment, this CaBP was identified as a monomer and its molecular weight was about 21 kDand isoelectric point (pI) value was about 4.1 using isoelectrofocusing. This CaBP was able to bind Ca2+ even in the pressence of an excess MgCl2 and KCI both in solution. In the SDS-PAGE, the Ca2+-bound form was slower than the Ca2+-free form in the nondenaturing PAGE. This means that the CaBP undergoes conformational change in the Ca2+-bound condition. Furthermore, UV absorption spectrum and fluorescence intensity of this CaBP was investigated. UV absorption peak was appeared at about 258 nm and decreased somewhat in Ca2+-bound condition. In the measurement of fluorescence, maximum intensity was appeared at 303 nm and decreased in Ca2+-bound state, similarly as UV absorption spectrum. These show distinct changes upon Ca2+-binding, which indicate of structural and/or dynamic changes largely reminiscent of other members of the EF-hand Ca2+-binding protein family.

• Membrane and Water Treatment
• /
• v.4 no.2
• /
• pp.109-126
• /
• 2013

Surface modification by low-pressure ammonia ($NH_3$) plasma on commercial thin-film composite (TFC) membranes was investigated in this study. Surface hydrophilicity, total surface free energy, ion exchange capacity (IEC) and zeta (${\zeta}$)-potentials were determined for the TFC membranes. Qualitative and quantitative analyses of the membrane surface chemistry were conducted by attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy. Results showed that the $NH_3$ plasma treatment increased the surface hydrophilicity, in particular at a plasma treatment time longer than 5 min at 50 W of plasma power. Total surface free energy was influenced by the basic polar components introduced by the $NH_3$ plasma, and isoelectric point (IEP) was shifted to higher pH region after the modification. A ten (10) min $NH_3$ plasma treatment at 90 W was found to be adequate for the TFC membrane modification, resulting in a membrane with better characteristics than the TFC membranes without the modification for water treatment. The thin-film chemistry (i.e., fully-aromatic and semi-aromatic nature in the interfacial polymerization) influenced the initial stage of plasma modification.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.11 no.2
• /
• pp.134-139
• /
• 2006

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) from Methylomicrobium sp. HG-1, which belongs to the type I group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in an ${\alpha}_2{\beta}_2$ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and $60^{\circ}C$, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.