• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.999-1006
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• 2015

The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The fulllength cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50℃, respectively. The Michaelis-Menten constant (K_m) and maximal velocity (V_max) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca²⁺, Cu²⁺, and Na⁺, and strongly inhibited by Pb²⁺ and Mn²⁺. The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.33-43
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• 2013

A bacterium producing a fibrinolytic enzyme was isolated from Cheonggukjang. The bacterium was identified as a strain of Bacillus amyloliquefaciens by 16S rDNA analysis and designated as B. amyloliquefaciens HC188. The optimum culture medium appeared to be one containing 0.5% (w/v) maltose and 0.5% (w/v) soytone. Bacterial growth in the optimal medium at $37^{\circ}C$ reached the stationary phase after 27 h of incubation and the fibrinolytic enzyme showed optimum activity at 24 h. The enzyme was purified by 20-80% ammonium sulfate precipitation, CM Sepharose fast flow ion exchange chromatography, and Sephacryl S-200HR column chromatography. Its specific activity was 38359.3 units/mg protein and the yield was 5.5% of the total activity of the crude extracts. The molecular weight was 24.7 kDa and the amino acids of the N-terminal sequence were AQSVPYGVSQIKAPA. The fibrinolytic enzyme activity had an optimum temperature of $40^{\circ}C$ and an optimum pH of 8.0, and the enzyme was stable in the ranges $20-40^{\circ}C$ and pH 6.0-8.0. Enzyme activity was increased by $Ca^{2+}$ and $Co^{2+}$ but inhibited by $Cu^{2+}$, EDTA, and PMSF. It is suggested that the purified enzyme is a metallo-serine protease.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.129-134
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• 1987

Aspergillus sp. (MK-24) producing a biological active substance that inhibited the venom proteinase activity was isolated from soil. The substance also inhibited the activity of trypsin and coagulation of blood, but did not inhibit papain, $\alpha$-chymotrypsin and pepsin. The substance was partially purified from culture filtrate by precipitaion with acetone, and by chromatography of DEAE-Sepadex A-50 column and Amberlite IRC-50 ion exchange. The inhibitory substance was stable in the wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but not stable at $65^{\circ}C$ in the alkaline pH. Only 12% of the activity was decreased by the heat treatment at 10$0^{\circ}C$ for two hours. The inhibition on venom proteinase (Agkistrodon bromohoffi brevicaudus) was a mixed type. The inhibitory activity depended on the preincubation time and completely depressed by cupric, zinc and cobalt ions. The inhibition on the venom proteinase was appeared strongly on casein but not on ovalbumin or hemoglobin as a substrate.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.133-148
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• 1987

Gibberellin(GA) 3-$\beta$ hydroxylation is very important for the shoot elogation in the higher plants, since only 3$\beta$-hydryoxylated GAs promote shoot elogation in several plants. Fluctuation of 3$\beta$-hydryoxylase activity was examined during seed maturation using two cultivars of , P. vulgaris, Kentucky Wonder (normal) and Masterpiece (dwarf). Very immature seeds of both cultivars contain high level of 3$\beta$-hydroxylase activity (per mg protein). Both cultivars showed maximum of enzyme activity (per seed) in the middle of their maturation process. Gibberellin 3$\beta$-hydroxylase catalyzing the hydroxylation of GA20 to GA1 was purified 313-fold from very early immature seeds of P. vulgaris. Crude soluble enzyme extracts were purified by 15% methanol precipitation, hydrophobic interaction chromatogrphy, DEAE ion exchange column chromatography and gel filtration HPLC. The 3$\beta$-hydroxylase activity was unstable and lost much of its activity duting the purification. The molecular weight of purified enzyme was extimated to be 42, 000 by gel filtration HPLC and SDS-PAGE. The enzyme exhibited maximum activity at pH 7.7. The Km values for [2.3-3H] GA20 and [2.3-3H]GA9 were 0.29 $\mu$M and 0.33 $\mu$M, respectively. The enzyme requires 2-oxoglutarate as a cosubstrate; the Km value for 2-oxoglutarate was 250 $\mu$M using 3H GA20 as a substrate. Fe2+ and ascorbate significantly activated the enzyme at all purification steps, while catalase and BSA activated the purified enzyme only. The enzyme was inhibited by divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Effects of several GAs and GA anaogues on the putrified 3$\beta$-hydroxylase were examined using [3H]GA9 and GA20 as a substrates. Among them, GA5, GA9, GA15, GA20 and GA44 inhibited the enzyme activity. [13C, 3H] GA20 was converted by the partially purified enzyme preparation to [13C, 3H]GA1, GA5 and GA6, which were identified by GC-MS, GA9 was converted only GA4, GA15 and GA44 were converted to GA37 and GA38, respectively. GA5 was epoxidized to GA6 by the preparation. This suggests that 3$\beta$-hydroxylation of GA20 and epoxidation of GA5 are catalyzed by the same enzyme in P, vulgaris.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.83-93
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• 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 Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.24 no.4
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• pp.519-534
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• 2019

When the fresh water from the artificial lakes (Ganwolho and Bunamho) were discharged to Cheonsu Bay in summer to prevent the flood over the reclaimed farmland near the lakes, the impact on water qualities (nutrients, organic matters, trace metals) within the bay was investigated through four surveys (June, July, August and October, 2011). Dissolved inorganic nitrogen (DIN) increased about as much as 3-4 times over the whole water column when the freshwater was discharged. And the main species composition of DIN changed from ammonia to nitrate. Dissolved inorganic phosphorus (DIP) decreased as much as 2 times in surface waters, but increased as much as 1.5 times in deep waters, and also silicate concentrations increased as much as 3-4 times in deep waters of the inner bay. The N/P ratios in Chunsu bay seawaters were much higher (2 to 7 times) than the Redfield ratio when the freshwaters were discharged, which indicated the phosphorus limiting in the phytoplankton growth. Dissolved organic carbon (DOC) and nitrogen (DON) increased as much as about 2 times. In addition, particulate organic matters (POC, PON, POP, Bio-Si) increased as much as above 2 times in the surface waters of the inner bay. Trace metals (Fe, Mn, Co, Ni, Cu) increased in the surface waters of the inner bay, but dissolved Cd concentrations decreased as much as 2 times. Therefore, when the contaminated fresh waters from the artificial lakes were discharged into the bay, nutrients, organic matters and trace metals generally increased compared to normal period. Since the phytoplankton bloom occurred in the surface waters of the inner bay, dissolved oxygens at the surface waters were oversaturated and hence hypoxic in the deep waters. Highly enriched nutrients concentrations were found in deep waters of the inner bay, which was accompanied with the hypoxic condition. Finally, the water quality in the inner bay of the Chunsu bay was deteriorated from less than grade 3 in normal periods to grade 5 when the freshwaters from the artificial lakes were discharged in summer.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Current Research on Agriculture and Life Sciences
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• v.4
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• pp.55-64
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• 1986

Polyphenol oxidase in japanese pear (Pyrus communis var. mansamkil) was isolated, partially purified and its some properties were investigated. Polyacrylamide disc gel electrophoresis indicated two bands with polyphenol oxidase activity in the extract from acetone dry powder of par flesh. These two polyphenol oxidases (PPO A and PPO B) were purified through acetone precipitation and diethylaminoethyl cellulose column chromatography. PPO A and B were purified 7.8 fold and 8.7 fold by the present procedure, respectively. The Rm values of partially purified PPO A and B were estimated to be 0.58 and 0.68, respectively. The optimum temp, and pH of PPO A activity were $33^{\circ}C$ and pH 7.0, while those of PPO B were $30^{\circ}C$ and pH 4.2, respectively. Two PPO were unstable over the temperature of $60^{\circ}C$. The substrate specificity of pear PPO showed high affinity toward o-diphenolic compounds, especially catechol in PPO A and chlorogenic acid in PPO B, but inactive toward m-diphenol, p-diphenol and monophenols. PPO A showed affinity toward the trihydroxyphenolic compound. $Zn^{{+}{+}}$ activated the PPO A activity but $Fe^{{+}{+}}$ inhibited PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity but $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$ and $Hg^{{+}{+}}$ inhibited at 10mM concentration. $Cu^{{+}{+}}$ activated the enzyme action at low concentrations but inhibited at high concentration. Inhibition studies indicated that L-ascorbic acid, L-cysteine and thiourea were most potent. The Km values of PPO A and PPO B for catechol were 20mM and 14.3mM, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.15 no.1
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• pp.1-25
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• 1982

Limnological study on the physico-chemical properties and biological characteristics of the Lake Ok-Jeong was made from May 1980 to August 1981. For the planktonic organisms in the lake, species composition, seasonal change and diurnal vertical distribution based on the monthly plankton samples were investigated in conjunction with the physico-chemical properties of the body of water in the lake. Analysis of temperature revealed that there were three distinctive periods in terms of vertical mixing of the water column. During the winter season (November-March) the vertical column was completely mixed, and no temperature gradient was observed. In February temperature of the whole column from the surface to the bottom was $3.5^{\circ}C$, which was the minimum value. With seasonal warming in spring, surface water forms thermoclines at the depth of 0-10 m from April to June. In summer (July-October) the surface mixing layer was deepened to form a strong thermocline at the depth of 15-25 m. At this time surface water reached up to $28.2^{\circ}C$ in August, accompanied by a significant increase in the temperature of bottom layer. Maximum bottom temperature was $r5^{\circ}C$ which occurred in September, thus showing that this lake keeps a significant turbulence Aehgh the hypolimnial layer. As autumn cooling proceeded summer stratification was destroyed from the end of October resulting in vertical mixing. In surface layer seasonal changes of pH were within the range from 6.8 in January to 9.0 in guutuost. Thighest value observed in August was mainly due to the photosynthetic activity of the phytoplankton. In the surface layer DO was always saturated throughout the year. Particularly in winter (January-April) the surface water was oversaturated (Max. 15.2 ppm in March). Vertical variation of DO was not remarkable, and bottom water was fairly well oxygenated. Transparency was closely related to the phytoplankton bloom. The highest value (4.6 m) was recorded in February when the primary production was low. During summer transparency decreased hand the lowest value (0.9 m) was recorded in August. It is mainly due to the dense blooming of gnabaena spiroides var. crassa in the surface layer. A. The amount of inorganic matters (Ca, Mg, Fe) reveals that Lake Ok-Jeong is classified as a soft-water lake. The amount of Cl, $NO_3-N$ and COD in 1981 was slightly higher than those in 1980. Heavy metals (Zn, Cu, Pb, Cd and Hg) were not detectable throughout the study period. During the study period 107 species of planktonic organisms representing 72 genera were identified. They include 12 species of Cyanophyta, 19 species of Bacillariophyta, 23 species of Chlorophyta, 14 species of Protozoa, 29 species of Rotifera, 4 species of Cladocera and 6 species of Copepoda. Bimodal blooming of phytoplankton was observed. A large blooming ($1,504\times10^3\;cells/l$ in October) was observed from July to October; a small blooming was present ($236\times10^3\;cells/l$ in February) from January to April. The dominant phytoplankton species include Melosira granulata, Anabaena spiroides, Asterionella gracillima and Microcystis aeruginota, which were classified into three seasonal groups : summer group, winter group and the whole year group. The sumner group includes Melosira granulate and Anabaena spiroides ; the winter group includes Asterionella gracillima and Synedra acus, S. ulna: the whole year group includes Microtystis aeruginosa and Ankistrodesmus falcatus. It is noted that M. granulate tends to aggregate in the bottom layer from January to August. The dominant zooplankters were Thermocpclops taihokuensis, Difflugia corona, Bosmina longirostris, Bosminopsis deitersi, Keratelle quadrata and Asplanchna priodonta. A single peak of zooplankton growth was observed and maximum zooplankton occurrence was present in July. Diurnal vertical migration was revealed by Microcystis aeruginosa, M. incerta, Anabaena spiroides, Melosira granulata, and Bosmina longirostris. Of these, M. granulata descends to the bottom and forms aggregation after sunset. B. longirostris shows fairly typical nocturnal migration. They ascends to the surface after sunset and disperse in the whole water column during night. Foully one species of fish representing 31 genera were collected. Of these 13 species including Pseudoperilnmpus uyekii and Coreoleuciscus splendidus were indigenous species of Korean inland waters. The indicator species of water quality determination include Microcystis aeruginosa, Melosira granulata, Asterionelta gracillima, Brachionus calyciflorus, Filinia longiseta, Conochiloides natans, Asplanchna priodonta, Difflugia corona, Eudorina elegans, Ceratium hirundinella, Bosmina longirostris, Bosminopsis deitersi, Heliodiaptomus kikuchii and Thermocyclops taihokuensis. These species have been known the indicator groups which are commonly found in the eutrophic lakes. Based on these planktonic indicators Lake Ok-Jeong can be classified into an eutrophic lake.

• Applied Biological Chemistry
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• v.14 no.1
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• pp.59-97
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• 1971

As a study on the cellulase of Myriococcum albomyces the culture media for enzyme formation and properties of its crude preparation were investigated and the crude enzyme preparation was further fractionated. The results are summarized as follows: 1. Wheat bran solid culture produced stronger activities of cellulase than rice bran or defatted soy bean meal solid culture. 2. Shaking culture with wheat bran, rice bran or defatted soy bean meal produced higher cellulase activities than solid culture with the corresponding media. 3. The enzyme formation was higher at $45^{\circ}C$ than at $37^{\circ}C$ or $50^{\circ}C$ regardless of the kind of culture medium. 4. The formation of CMCase activity was more promoted by organic nitrogen source than inorganic nitrogen source. 5. The formation of cellulase activities were increased 1.5 to 3.0-fold by adding CMC, Avicel or cellulose powder as an inducer into 5% wheat bran basal medium. 6. Cellulase production using a tank culture procedure with addition of CMC or Avicel as an inducer was the highest at fifth day and thereafter decreased slightly. 7. The crude enzyme preparation showed pH optimum in 4.0 to 4.5, and pH stability in the range of 3.5 to 8.0. Optimum temperature for the activity was $65^{\circ}C$ which was higher than among other cellulases and it was stable at $60^{\circ}C$ for 120 minutes. 8. Dialyzed crude enzyme was activated by $Ca^{++}$ and $Mg^{++}$, but inhibited by $Hg^{++}$, $Cu^{++}$ and $Ag^{+}$. 9. Four different types of cellulase, i. e., fraction I, fraction II-a, fraction II-b, and fraction III were purified from the culture filtrate of Myriococcum albomyces through a sequence of ammonium sulfate fractionation, and elution chromatography on DEAE-Sephadex A-25, Amberlite CG-25 type 2 and hydroxyapatite columns. 10. These four cellulase fractions were showed to be homogenous by electrophoresis and ultracentrifugation and also gave a typical ultraviolet absorption spectrum of protein. 11. Four purified fraction showed different specificity toward substrates, fraction I has a stronger activity toward Avicel, cellulose powder, and gauze than that of other cellulase fractions. Fraction II-a had a powerful activity toward cellobiose but it was almost inactive agaisnt fibrous cellulose contrary to fraction I. On the contrary, the main component fraction II-b had a fairly higher activity on CMC and Avicel. Activity of fraction II-b toward cellobiose was about one-third of that of fraction II-a and activity on Avicel was lower than that of fraction I. Fraction III had a more powerful activity in decreasing viscosity of CMC. 12. Final hydrolysis products of fibrous cellulose by each fraction were cellobiose and glucose. Whereas oligosaccharides were predominant in the early stage of hydrolysis, prolonged reaction produced more glucose than cellobiose. Fraction I and fraction II-a acted synergically on Avicel. 13. Optimum pH for the activities of cellulase fraction I, fraction II-a, fraction II-b and fraction III were found to be 5.5, 5.0, 4.0 and $4.0{\sim}4.5$, respectively. These fractions were found to be stable in the range of pH $3.0{\sim}7.5$. 14. Optimum temperature for the activities of fraction I, fraction II-a, fraction II-b, and fraction III were $50^{\circ}C$, $55^{\circ}C$, $60^{\circ}C$ and $55^{\circ}C$, respectively. No less of activity was found by heating 120 minutes at $55^{\circ}C$ and fraction II-a was more stable than the others at $60^{\circ}C$. 15. Fraction I and fraction II-b were activated by $Ca^{++}$ and $Mg^{++}$ but inhibited by $Hg^{++}$ and $Ag^{+}$.