• Journal of Environmental Health Sciences
• /
• v.18 no.1
• /
• pp.84-94
• /
• 1992

In an attempt to develop the immobilized biocatalysts based on immobilized Saccharomyces cerevisiae, immobilized yeast was investigated with respect to the conditions affected to ethanol productivities. Saccharomyces cerevisiae was immobilized in the form of the beads by magnetic-calcium alginate, non magnetic-calcium alginate and acrylamide polymerization. Magnetic immobilized yeast, nonmagnetic immobilized yeast and polyacrylamide immobilized yeast were compared in respect of their pH stability, thermostability, heat tolerance, the relation between the concetration of native yeast and retained activity of immobilized yeast, the activity depending on bead size of immobilized yeast, and the effects of magnesium and cobalt on the activities. The more small bead had retained the higher activity for the three kinds of immobilized yeast. In case of 1.0mm diameter of beads, the retained activity was 40~50% for the all groups. The pH stability profile for the immobilized yeast showed a broad range of optimun activity while the native yeast gave a sharp pick for its optimun pH value. The thermostability was at the range of 25~55$^{\circ}$C for the immobilized yeast groups. It was investigated that the influent magnesium and cobalt concentration, and the relative activity have an influent on heat tolerance at steady state. Both protein content released from immobilized yeast and activity of immobilized yeast were changed after activation of immobilized yeast cell.

• Journal of Life Science
• /
• v.8 no.6
• /
• pp.627-637
• /
• 1998

In order to utilize tuna pyloric caeca among fish intestines wasted when treated raw fish in fish processing manufactory, a crude enzyme with high proteolytic activity was extracted and its optimum condition were investigated. An immobilized enzymes also were prepared by adsorption method to enhance thermostability of the crude proteinase. The yield of the crude proteinase was approximately 2.7% on dry basis. The proteolytic activity for casein was 0.54 U/mg protein, for BTEE 1.10 U/mg protein, and for BAEE 2.69 U/mg protein. It was almost similar to that of the commercial trypsin purified. Optimum hydrolysis activity of the crude proteinase was about 80%, as the degree of hydrolysis for casein, at pH 10.0 and 45$^{\circ}C$ for 12 hrs. Also, when the crude proteinase was immobilized on DEAE-Cellulose and chitin, the residual activities remained after 7 days of pre-incubation time were maintained about 90% or more and their thermostabilities were enhanced by about 50%, compared with the native enzyme.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.22 no.2
• /
• pp.83-93
• /
• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• The Korean Journal of Food And Nutrition
• /
• v.1 no.1
• /
• pp.25-32
• /
• 1988

Properties of purified isocitrate lyase from Saccharomycopsis lipolytica ATCC 44601 and MX9-11RX8 temperature-sensitive mutant were investigated. Purified isocitrate lyase from temperature-sensitive mutant was indistinguishable from the wild type enzyme with respect to the isoelectric pH(5.3), the thermostability and Km value for threo-Ds-Isocitrate(about 0.2 mM). When isocitrate lyase induced by acetate minimal medium at 33$^{\circ}C$, MX9-11RX8 mutant did not express enzyme activity but did synthesize polypeptide chain whose electrophoretic mobilities were equal to those of the purified enzymes.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2005.06a
• /
• pp.283-284
• /
• 2005

• Journal of Microbiology
• /
• v.39 no.4
• /
• pp.245-249
• /
• 2001

• Journal of Microbiology and Biotechnology
• /
• v.4 no.2
• /
• pp.113-118
• /
• 1994

A bacterial strain NS-83 isolated from soil was able to produce an extracellular thermostable protease. The strain was identified as Pseudomonas aeruginosa based on its morphological and physiological characteristics. A thermostable protease from this strain has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification procedures included hydrophobic interaction, ion exchange, and gel filtration chromatography. The $M_r$ and the pl of the enzyme were 32,000 and 5.9, respectively. The optimal pH at 55$^{\circ}C$ and the optimal temperature at pH 7.0 were 8.0 and 60$^{\circ}C$, respectively. The D-values of the enzyme at 60, 65, and 70$^{\circ}C$ were 22, 2.1, and 0.75 hrs, respectively. The enzyme activity was significantly inhibited in the presence of 1 mM o-phenanthroline or EDTA, suggesting that the enzyme is metalloprotease. The $K_m$, and $V_{max}$ for Hammarsten casein were found to be 3.2 mg/ml and 0.918 unit/ml, respectively. These enzymatic properties were similar to those of elastase produced from P. aeruginosa IFO 3455, but the enzyme was clearly different from the reported elastase, in respect to $Ca^{++}$ effects on enzyme-thermostability. This property, together with amino acid composition analysis, confirmed that the enzyme differs from the known P. aeruginosa elastase.

• BMB Reports
• /
• v.38 no.1
• /
• pp.17-23
• /
• 2005

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

• BMB Reports
• /
• v.39 no.1
• /
• pp.105-110
• /
• 2006

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as $90^{\circ}C$. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of $90^{\circ}C$. When using xylan from birchwood as substrate, it exhibits $K_m$ and $V_{max}$ values of $2.6{\pm}0.6\;mg/ml$ and $428{\pm}26\;U/mg$, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to $70^{\circ}C$. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at $70^{\circ}C$ for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.5
• /
• pp.639-647
• /
• 2014

Laccases have a broad range of industrial applications. In this study, we immobilized laccase on $SiO_2$ nanoparticles to overcome problems associated with stability and reusability of the free enzyme. Among different reagents used to functionally activate the nanoparticles, glutaraldehyde was found to be the most effective for immobilization. Optimization of the immobilization pH, temperature, enzyme loading, and incubation period led to a maximum immobilization yield of 75.8% and an immobilization efficiency of 92.9%. The optimum pH and temperature for immobilized laccase were 3.5 and $45^{\circ}C$, respectively, which differed from the values of pH 3.0 and $40^{\circ}C$ obtained for the free enzyme. Immobilized laccase retained high residual activities over a broad range of pH and temperature. The kinetic parameter $V_{max}$ was slightly reduced from 1,890 to 1,630 ${\mu}mol/min/mg$ protein, and $K_m$ was increased from 29.3 to 45.6. The thermal stability of immobilized laccase was significantly higher than that of the free enzyme, with a half-life 11- and 18-fold higher at temperatures of $50^{\circ}C$ and $60^{\circ}C$, respectively. In addition, residual activity was 82.6% after 10 cycles of use. Thus, laccase immobilized on $SiO_2$ nanoparticles functionally activated with glutaraldehyde has broad pH and temperature ranges, thermostability, and high reusability compared with the free enzyme. It constitutes a notably efficient system for biotechnological applications.