• Journal of Microbiology and Biotechnology
• /
• v.8 no.4
• /
• pp.353-360
• /
• 1998

A bacterial strain PN33 producing large amounts of extracellular pectin lyase (PNL, EC 4.2.2.10) was isolated from soil. The isolated bacterium was identified as a strain of Bacillus sp. Production of PNL by the strain was induced only by pectins, with a higher degree of esterification, which had been added to the culture medium as a sole carbon source. The optimal medium for PNL production was determined to consist of 10 g pectin, 2 g yeast extract, 4 g $K_2HPO_4{\cdot}3H_2O$, 0.6 g $MgSO_4$, and 0.11 g $CaCl_2$ per liter (pH 7.0). The PNL activity in the culture supernatant reached the highest level of 132 mU/ml after 32 h cultivation at $37^{\circ}C$ in the optimal medium. The PNL produced was purified to homogeneity by ammonium sulfate fractionation (50~80%), and cation exchange and size exclusion chromatographies. The molecular mass of the enzyme was estimated to be approximately 52 kDa by SDS-PAGE. Almost the same mass was determined by nondenaturing PAGE, indicating that the functional enzyme had a monomeric structure. As expected, the PNL exhibited higher activities on the highly esterified pectins whereas it gave no detectable activity on polygalacturonic acid. The enzyme showed the highest activity at the acidic pH of 6.0, exceptional for a bacterial PNL. Maximum activity was measured at $40^{\circ}C$, although the stability f the purified enzyme was poor at this temperature. alcium (1 mM) was found to activate the PNL activity by $50\%$, and also remarkably increased the thermal stability f the enzyme. Phenylmethylsulfonylfluoride (PMSF) and iethylpyrocarbonate (DEPC) inhibited the PNL activity lmost completely at the concentration of 5 mM. This result ndicates that some serine and histidine residues of the nzyme may play an essential role for catalytic function of he enzyme.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.9
• /
• pp.1237-1244
• /
• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.11
• /
• pp.1540-1548
• /
• 2012

Manganese peroxidase (MnP) was isolated from the culture filtrate of the wood log mushroom Stereum ostrea (S. ostrea), grown on Koroljova medium, and then purified by ammonium sulfate [70% (w/v)] fractionation, DEAE-cellulose anion exchange chromatography, and Sephadex G-100 column chromatography, with an attainment of 88.6-fold purification and the recovery of 22.8% of initial activity. According to SDS-PAGE the molecular mass of the MnP was 40 kDa. The optimal pH and temperature were found to be 4.5 and $35^{\circ}C$, respectively. The enzyme was stable even after exposure to a pH range of 4.5 to 6.0, and at temperatures of up to $35^{\circ}C$ at a pH of 4.5 for 1h. The $K_m$ and $V_{max}$ values for the substrate phenol red were found to be $8{\mu}m$ and 111.14 U/mg of protein, respectively. The MnP also oxidized other substrates such as guaiacol, DMP, and veratryl alcohol. Sodium azide, EDTA, SDS, $Cu^{2+}$, and $Fe^{2+}$, at 1-5 mM, strongly inhibited enzyme activity, whereas $Ca^{2+}$ and $Zn^{2+}$ increased enzyme activity. The participation of the purified enzyme in the decolorization of dyes suggests that S. ostrea manganese peroxidase could be effectively employed in textile industries.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.11
• /
• pp.1536-1543
• /
• 2013

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at $28^{\circ}C$ in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in $67.9{\pm}0.64%$ mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

• Journal of Microbiology
• /
• v.35 no.2
• /
• pp.109-116
• /
• 1997

An extracellular proteinase of Candida albicans was purified by a combination of 0~75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45$^{\circ}C$. The addition of divalent cations, $Ca^{2+}$, Zn$^{2+}$ and $Mg^{2+}$, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe$^{2+}$, Ag$^{2+}$ and Cu$^{2+}$. With BSA as substrate, an apparent $K_m$ was determined to be 7$\times$10$^{-7}$ M and $K_i$, using pepstatin A as an inhibitor, was 8.05$\times$10$^{-8}$ M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P$_1$ position, but the enzyme activity was highly reduced when the P$_2$ position was phe or pro. This enzyme showed antigenicity against sera of patients with candidiasis.

• Korean Journal of Agricultural Science
• /
• v.46 no.4
• /
• pp.907-914
• /
• 2019

Ammonia (NH₃) that reacts with nitric or sulfuric acid in the air is the major culprit contributing to the formation of fine particulate matter (PM_2.5). NH₃ volatilization mainly originates from nitrogen fertilizer and livestock manure applied to arable soil. Cation exchange capacity (CEC) of peat moss (PM) and zeolite (ZL) is high enough to adsorb ammonium (NH₄⁺) in soil. Therefore, they might inhibit volatilization of NH₃. The objective of this study was to compare the effect of PM and ZL on NH₃ volatilization from upland soil. For this, a laboratory experiment was carried out, and NH₃ volatilization from the soil was monitored for 12 days. PM and ZL were added at the rate of 0, 1, 2, and 4% (wt wt^-1) with 354 N g m^-2 of urea. Cumulative NH₃-N volatilization decreased with increasing addition rate of both materials. Mean value of cumulative NH₃-N volatilization across application rate with PM was lower than that with ZL. CEC increased with increasing addition rate of both materials. While the soil pH increased with ZL, it decreased with PM. Increase in CEC resulted in NH₄⁺ adsorption on the negative charge of the external surface of both materials. In addition, decrease in soil pH hinders the conversion of NH₄⁺ to NH₃. Based on the above results, the addition of PM or ZL could be an optimum management to reduce NH₃ volatilization from the soil. However, PM was more effective in decreasing NH₃ volatilization than ZL due to the combined effect of CEC and pH.

• KSBB Journal
• /
• v.29 no.3
• /
• pp.131-138
• /
• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.

• KSBB Journal
• /
• v.9 no.1
• /
• pp.55-62
• /
• 1994

Glucose oxidase(EC 1.1.3.4) was purified to electrophoretic homogeneity from Aspergillus niger by a combination of ammonium sulfate fractionation, ion exchange chromatography, and ultrafiltration. Two active fractions A and B, of glucose oxidase were obtained from the hydrophobic chromatography on phenyl sepharose CL-4B. The enzyme A and B were glycoproteins with the same denatured molecular weight of 78, 000 and had specific activities of 2, 191 and 1, 273-units/mg proteins, respectively. But the two enzymes showed differences in native molecular weight that was measured by HPLC gel filteration, maximum absorbtion wavelength and isoelectric point. The enzyme A oxidized $\beta$-D-glucose only and was resistant to sodium dodecyl sulfate. Activity optimum was found at $30^{\circ}C$ and pH 3.5. Also the enzyme A was inhibited greatly by $Hg^{2+}$(10mM). The results of chemical modification experiments suggested that cysteine and cystine residues might be involved in the active site of the enzyme A.

• BMB Reports
• /
• v.40 no.3
• /
• pp.358-367
• /
• 2007

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 $\mu$g/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 $\pm$ 6.8), Bre-04 (41.5 $\pm$ 4.8), and Lu-04 (11.4 $\pm$ 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with $TC_50$ and $EC_50$ of 5.176 mg/ml and 3.054 $\mu$g/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.

• Journal of the Korean Society of Tobacco Science
• /
• v.21 no.2
• /
• pp.160-170
• /
• 1999

Purification and biochemical experiments on the carboxylesterases-III (CE-III) from the indian meal moth, Plodia interpunctella (Hubner) were carried out to understand their enzymemological characteristics. The CE-III from the fifth instar larvae was purified by means of ammonium sulfate fractionation, gel permeation choromatography and ion exchange choromatography. The optimal temperature for the reaction of the CE-III on the 4 substrates ($\alpha$-Na, $\alpha$-Nb, $\beta$-Na and $\beta$-Nb) was confirmed at 4$0^{\circ}C$. The optimal pH for the reactions on the substrates $\alpha$-Na and $\alpha$-Nb was 7.5. But the optimal pH on the substrate $\beta$-Na and $\beta$-Nb was 8.0. The optimal substrate concentration for the reactions of the CE-III was 3.16 X 10$^{-3}$ M in $\alpha$-Na and $\beta$-Nb. On the substrate $\beta$-Na and $\alpha$-Nb, the optimal substrate concentration was 1.0 X 10$^{-3}$ M for CE-III. The $V_{max}$ and $K_{m}$ values of the carboxylesterases were varied by the substrates as followings: the $V_{max}$ of CE-III was 45.9 for $\alpha$-Na, 52.6 for $\beta$-Na, 36.4 for $\alpha$-Nb, and 83.3 ($\mu$ mol/min/mg protein) for $\beta$-Nb. The $K_{m}$ of CE-III was 1.43 X 10$^{-4}$ M for $\alpha$-Na, 3.57 x 10$^{-5}$ M for $\beta$-Na, 9.17 X 10$^{-5}$ M for $\alpha$-Nb, and 7.14 X 10$^{-5}$ M for $\beta$ -Nb, respectively. The CE-III seemed to have somewhat high thermostability considering that the temperature for effective denaturation on activity was about 5$0^{\circ}C$ ～ 6$0^{\circ}C$.EX>.EX>.