• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.757-764
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• 2018

A cellulolytic fungus, YDJ14, was isolated from compost and identified as an Aspergillus sp. strain. Three extracellular ${\beta}$-glucosidases, BGL-A1, BGL-A2, and BGL-A3, were separated using ultrafiltration, ammonium sulfate fractionation, and High-Q chromatography. The molecular masses of the three enzymes were estimated to be 100, 45, and 40 kDa, respectively, by SDS-PAGE. The optimum pH and temperature of BGL-A3 were 5.0 and $50^{\circ}C$, respectively, whereas the optimum pH and temperature of BGL-A1 and BGL-A2 were identical (4.0 and $60^{\circ}C$, respectively). The half-life of BGL-A3 at $70^{\circ}C$ (2.8 min) was shorter than that of BGL-A1 and BGL-A2 (12.1 and 8.8 min, respectively). All three enzymes preferred p-nitrophenyl-${\beta}$-$\text\tiny{D}$-glucopyranoside (pNPG) and hardly hydrolyzed cellobiose, suggesting that these enzymes were aryl ${\beta}$-glucosidases. The $K_m$ of BGL-A3 (1.26 mM) for pNPG was much higher than that of BGL-A1 and BGL-A2 (0.25 and 0.27 mM, respectively). These results suggested that BGL-A1 and BGL-A2 were similar in their enzymatic properties, whereas BGL-A3 differed from the two enzymes. When tilianin (a flavone glycoside of acacetin) was reacted with the three enzymes, the inhibitory activity for monoamine oxidase, a target in the treatment of neurological disorders, was similar to that shown by acacetin. We conclude that these enzymes may be useful in the hydrolysis of flavone glycosides to improve their inhibitory activities.

• Applied Biological Chemistry
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• v.39 no.1
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• pp.60-66
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• 1996

To investigate a possible application of three strains of lactic acid bacteria(strain No. 49. No. 61. No. 75) from kimchi in milk fermentation industry, the optimal condition for production of intracellular ${\beta}-galactosidase$ from Lactobacillus(L.) plantarum and its enzymatic properties were examined. The preferable carbon source of the medium for strain No. 49 in production of ${\beta}-galactosidase$ was MRS broth with 1.0% lactose instead of dextrose of pH 65. for strain No. 75 with 1.0% galactose and for strain No. 61 with 3.0% lactose at pH 7.5, respectively. The maximum enzyme production from strain No. 49, No. 75 was observed after 48 hours culture at $30^{\circ}C$ in a medium containing the appropriate carbon source, from strain No. 61 after 48 hours culture at room temperature. The optimum temperature for ${\beta}-galactosidase$ activity from L. plantarum was $60^{\circ}C$ for strain No. 49, $37^{\circ}C$ for strain No. 61 and $50^{\circ}C$ for strain No. 75, respectively. The heat stability of enzyme activities for all three strains remained 90% at $45^{\circ}C$. The optimal pH was pH 6.5 and enzyme activities were most stable at pH for all three bacteria.

• Journal of Applied Biological Chemistry
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• v.52 no.4
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• pp.157-162
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• 2009

An agar-liquefying bacteria (SC-22), which produces a diffusible agarase that caused agar softening around the colony was isolated from Daecheong lake in Korea. Chemotaxanomic and phylogenetic analyses based on 16S rRNA gene sequences revealed the strain was classified as Cellvibrio mixtus SC-22. The isolate SC-22 showed maximal extracellular agarase activity with 58.5 U/mL after 48 h cultivation in the presence of 0.2% agar. It was observed that the isolate produced two kinds of extracellular and three kinds of intracellular isoenzymes. The major agarase was purified from the culture filtrate of agarolytic bacteria by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The molecular mass of the purified enzyme was estimated to be 25 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 7.0 and $50^{\circ}C$, respectively. The agarase activity was activated by $Fe^{2+}$, $Na^+$ and $Ca^{2+}$ ions while it was inhibited by $Hg^{2+}$, $Mn^{2+}$ and $Cu^{2+}$ at 1 mM concentration. The predominant hydrolysis product of agarose by the enzyme was galactose and disaccharide on TLC, indicating the cleavage of $\beta$-1,4 linkage in a random manner. The enzyme showed high substrate specificity for only agar and agarose among various polysaccharides.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.348-355
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• 2012

In the present study, we investigated the overexpression and characterization of bovine pancreatic (bp)- DNase I in Saccharomyces cerevisiae and Pichia pastoris. The bp-DNase I gene was fused in frame with the GAL10 promoter, $MF{\alpha}$, and GAL7 terminator sequences, resulting in the plasmid, pGAL-$MF{\alpha}$-DNaseI (6.4 kb). Also, the bp-DNase I gene was fused in frame with the AOX1 promoter, $MF{\alpha}$, and AOX1 terminator sequences, resulting in the plasmid, pPEXI (8.8 kb). The recombinant plasmids, pGAL-$MF{\alpha}$-DNaseI and pPEXI were introduced into S. cerevisiae and P. pastoris host cells, respectively. When the transformed yeast cells were cultured at $30^{\circ}C$ for 48 h in galactose or methanol medium, bp-DNase I was overexpressed and the most of activity was found in the extracellular fraction. P. pastoris transformant activity showed 45.5 unit/mL in the culture medium at 48 h cultivation, whereas S. cerevisiae transformant revealed 37.7 unit/mL in the extracellular fraction at 48 h cultivation. The enzymatic characteristics, such as DNA cleavage and half life were investigated. Treatment of the recombinant DNase I from P. pastoris induced degradation of the calf thymus DNA within 1 minute, and this DNA degradation rate was higher than that of commercial bp-DNase I (SIGMA) and the recombinant DNase I from S. cerevisiae.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.1
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• pp.37-45
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• 2013

This study was conducted to improve the yield of extracts from Alaska pollock Theragra chalcogramma head and sea tangle Laminaria japonica byproducts using various commercial enzymes, such as Alcalase, Flavourzyme, Neutrase (NH), and Protamex. Among the enzymatic hydrolysates, the yield was highest in hydrolysate incubated with NH for 4 h. NH-treated hydrolysates (NHH) also improved functional properties, such as angiotensin-I converting enzyme (ACE) inhibitory activity and 2,2-diphenyl-1-picryldrazyl (DPPH) radical scavenging activity, as compared to extracts from Alaska pollock head and sea tangle byproducts. Total free amino acid and taste values of NHH were 379.7 mg/100 mL and 24.03, respectively, after digestion for 4 h. These values are 2.2-fold and 1.9-fold higher compared with those of water soluble fractions extracted from Alaska pollock head and non-forming sea tangle, respectively. According to the taste value results, the major taste-active compounds among free amino acids of NHH were glutamic acid and aspartic acid. These results suggest that NHH can be used as an ingredient for natural seasoning preparation.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.126-130
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• 1989

In 14 kinds of ginsenosides in ginseng saponin, ginsenoside Rbr is contained the most abundantly. But ginsenoside Rd which is similar to ginsenoside R $b_1$in structure, was known to be superior to ginsenoside R $b_1$pharmaceutically. The convertible enzyme which can transform ginsenoside R $b_1$to Binsenoside Rd specifically among ginseng saponin, was purified homogeneously from Rhizopus japonicus. The optimal pH for the action of the enzyme was pH 4.8 to 5.0, and optimal temperature was 45$^{\circ}C$. The enzyme was stable in the range of pH 4.0 to 9.0, and the half activity of enzyme was remained by the thermal treatment at 6$0^{\circ}C$ for 2 hours. The enzyme activity was enhanced by addition of M $n^{++}$ or Fe, though inhibited by EDTA or o-phenanthroline. On the substrate specificity, the enzyme was. able to hydrolyze gentiobiose, cellobiose, amygdalin and prunasin, but not to hydrolyze any other kinds of Binsenosides besides Binsenoside R $b_1$. Km values of the enzyme for ginsenoside R $b_1$, gentiobiose and amygdalin were 5.0mM, 4.8mM and 3.7mM, respectively.3.7mM, respectively.y.

• Journal of Nutrition and Health
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• v.6 no.3
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• pp.1-8
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• 1973

Metabolic responses to the protein-free, high-carbohydrate diet and subsequent food restriction on the same diet at the level of 50% and 75% has been studied on the adult albino rats. The energy source was either corn starch or sugar. In experiment I, adult male rats weighing $509{\pm}8g$ were divided into two groups 10 rats each. Rats fed on the stock diet served as a control. Rats of restriction group received a protein free diet until they reduced their weight down to 400g and continue on a protein-calorie restriction diet until they reduced their weight down to 300g. In experiment II, 28 adult male rats and the same numbers of female rats weighing $329{\pm}5g$ and $223{\pm}4g$ respectively were divided into four groups, 7 males and females in each. Rats fed on a stock diet were sacrificed at the point when others started a protein free diet. These were served as the control. The protein free group received a protein free diet ad libitum for 4 weeks. The 50% restriction group and 75% restriction group were fed on a protein free diet coupled with food restriction at levels of 50% and 75% respectively for 3 weeks. In the result of this study: 1. The rate of body weight changes was similar between the males and the females. Feeding protein free diet ad lib. initiated a rapid weight lost of approximately 25% and protein free diet coupled with food restriction showed 37-43% reduction of their initial weight. 2. There was no significant differences in the value of the N concentration in liver, spleen, brain and muscle between controls and experimental groups. 3. Rats fed on protein free diet showed 1/10 value of the control in the nitrogen excretion in urine. However female showed less N excretion than male. 4. Observing blood picture, the effects of protein depletion and calorie restriction were not appeared any remarkable changes. 5. There was no sign of fatty liver which might result from protein depletion and calorie restriction. 6. Following semi-starvation, FAO and HMP-DH total enzyme activity was reduced, but activity per unit weight was relatively stable.

• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.330-339
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• 1997

Botrytis cinerea T91-1 has shown to produce at least four different polygalacturonases in a liquid medium containing citrus pectin as a carbon source. One of the enzymes, its molecular weight was estimated as 37 kDa by denatured polyacrylamide gel electrophoresis, was purified by a series of procedures including acetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. By viscometric analysis, the enzyme was revealed as an endo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Co^{2+}$, and $Cu^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was $55^{\circ}C$ and the enzyme showed optimal pH values between 4.0 and 4.5. The enzyme was stable up to 12 hours in the range of pH 4 to 7 and at the temperature below $30^{\circ}C$. Amino acid sequence from N-terminal up to 6 amino acids determined by Edman degradation showed little homology with polygalacturonases from fungi and plants.

• Korean Journal of Ichthyology
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• v.22 no.1
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• pp.1-8
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• 2010

We determined oxidative stress caused by thermal stress in olive flounder Paralichthys olivaceus based on the altered-mRNA expression and enzymatic activity of two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), along with monitoring of several other biomarkers. When the fish were exposed to acute thermal change (from $20^{\circ}C$ to $25^{\circ}C$ and $30^{\circ}C$), the expression and activity of both enzymes were significantly higher at elevated temperatures ($25^{\circ}C$ and $30^{\circ}C$) than at $20^{\circ}C$. Lipid peroxidation (LPO) was also higher at $25^{\circ}C$ and $30^{\circ}C$ than at $20^{\circ}C$. In addition, the plasma $H_2O_2$ concentration was significantly increased by thermal stress. Furthermore, we investigated changes due to thermal stress by measuring levels of plasma alanine aminotransferase (AlaAT) and aspartate aminotrasferase (AspAT). Both were significantly increased by thermal stress. As an immune indicator, the lysozyme concentration was lower at $30^{\circ}C$ than at $20^{\circ}C$, indicating that thermal stress decreases immune function. Therefore, thermal stress could induce oxidative stress and suppress immune function and can cause physiological stress.