• Journal of Microbiology and Biotechnology
• /
• v.10 no.5
• /
• pp.677-684
• /
• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Microbiology and Biotechnology Letters
• /
• v.32 no.3
• /
• pp.230-237
• /
• 2004

In this study nitroreductase from Pseudomonas sp. HK-6 capable of degrading 2,4,6-trinitrotoluene (TNT) was characterized. Through a series of purification process including ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose, three different fractions I, II, and III having the enzyme activity of NTRs whose molecular weights were approximately 27 kDa were detected in fractions from HK-6 cells. Specific activity of the three fractions were approximately 4.85 unit/mg, 5.47 unit/mg, and 5.01 unit/mg, and concentrated to 9.0-, 10.1-, and 9.3-fold compared to crude extract, respectively. The optimal pH and temperature for the three NTR fractions were approximately 7.5 and $30^{\circ}C$, respectively. Metal ions, $Ag^{＋}$ , $Cu^{ 2+}$, $Hg^{2＋}$ inhibited approximately 70% of enzymes activities of all NTR, while $Fe^{2＋}$ did not stimulate or inhibit the activities. Monitoring the effect of chemicals on the enzyme activity revealed that those NTR fractions lost enzyme activity in presence of $\beta$-mercaptoethanol, but were a little influenced by dithiothreitol, EDTA and NaCl. The three NTR fractions demonstrated enzyme activities for nitrobenzene and RDX as well as TNT.

• Korean Chemical Engineering Research
• /
• v.49 no.6
• /
• pp.840-845
• /
• 2011

Degradation products of agarose are biologically active and thus used as an ingredient in pharmaceuticals or functional cosmetics. Furthermore, it has been strongly considered as a substrate for bio-ethanol fermentation. Recently, we isolated new agarase-producing microorganism, Pseudoalteromonas sp. from south sea of Korea. In this study, we aimed to separate and purify the agarase from culture broth of this strain. Separation of agarase was performed by ion- exchange chromatography on DEAE-Sepharose resin. Equilibrium pH and volume ratio of resin to the amount of protein were optimized for the efficient adsorption of protein. 410 ${\mu}g$ of protein was completely adsorbed to 3 mL of resin at pH 7.5. The total amount of eluted protein increased as NaCl concentration increased to 400 mM at isocratic elution. Agarase was separated by linear gradient elution of NaCl (0~1,000 mM). Three major protein peaks were observed and the presence or absence of agarase in these eluted proteins was measured by Lugol's staining technique. Only six eluted protein fractions showed strong agarase activity.

• Microbiology and Biotechnology Letters
• /
• v.18 no.5
• /
• pp.490-495
• /
• 1990

The extracellular inulinase from Bacillus spp. was purified to a single protein through a sequence of operations including ammonium sulfate fractionation, heat treatment, DEAE Sepharose C1-6B ion exchange chromatography, Sephadex 6-100 and Sephadex 6-150 gel filtration. The purified enzyme was confirmed to be a $\beta$ -D-fructofuranosidase(EC 3.2.1.26) which was much more active on sucrose than on inulin(I/S = 0.2). The maximal inulinase activity was observed at pH 6.0 and at the temperature of $50^{\circ}C$. The mo1ecular weight of the enzyme was about 56, 000. Tryptophan and histidine residues of the enzyme molecule were found to be essential for its catalytic activity.

• Journal of Microbiology
• /
• v.42 no.2
• /
• pp.152-155
• /
• 2004

Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using dif-ferent aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the puri-fication of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (${\alpha}$ subunit and ${\beta}$ subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90％ sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a het-erodimer (${\alpha}$$_1$${\beta}$$_1$). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 $^{\circ}C$. PCR amplification of these two subunits of PCD4,5 revealed that the ${\alpha}$ subunit and ${\beta}$ subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.

• Microbiology and Biotechnology Letters
• /
• v.23 no.5
• /
• pp.506-512
• /
• 1995

A cell aggregation factor produced by Aspergillus sp. LAM 94-142 was purified and partially characterized. The factor was purified about 15 folds from culture broth by IRA 420 and IRC 120 treatment, 1% NaCl added acetone precipitation, and Sepharose 4B column chromatography with overall yield of 48%. It was heteropolysaccharide consisted of mannose, arabinose, and glucose with a molar ratio, 31:17:2, and its molecular weight was estimated to be about 900,000 daltons by Sepharodse 4B gel filtration method. The optimum pH and temperature was 8 and 40$\circ$C, respectively. The factor was stable in pH range of 3-9 and at 100$\circ$C for 90 min. The cell aggregation activity of the factor was inhibited by the addition of Hg$^{2+}$, Fe$^{2+}$, Cu$^{2+}$, and some polypeptides such as milk casein or hemoglobin. The factor aggregated Bacillus subtilis, B. macerans, B. turingiensis, E. coli, Peudomonas aeruginosa, P. fluorescens, P. malophilia, and weakly aggregated Staphylococcus sp., Sarcina lutea, P. putida and Cryptococcus neoformnans, but it didn't aggregate various strains of Candida sp. and Saccharomyces sp.

• Microbiology and Biotechnology Letters
• /
• v.18 no.4
• /
• pp.376-382
• /
• 1990

An extracellular cellulase producing bacterium YE-5 was isolated from soil, and identified as a Cellulomonas sp. by its taxonomical characteristics. The maximal activities of avicelase (0.35 units/ml), CMCase (3.18 units/ml), FPase (0.315 units/ml) and $\beta$-glucosidase (0.882 units/ml) were obtained when this strain was cultured for 48 hrs at $30^{\circ}C$ in a medium containing 0.8% (w/v) Solka floc, 0.06010 (wlv) urea, 0.1% (w/v) $K_2HP0_4$, 0.1% (w/v) $MgS0_4.7H2_0$, 0.2% (w/v) bacto peptone, 0.2% (w/v) yeast extract and pH 6.5. The cellulase was purified by ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex 6-100 column chromatography from culture filtrate of Cellulomonus sp. YE-5. The molecular weights of purified avieelase, CMCase I, and CMCase II were estimated to be about 95,000 ~ 105,000, 46,000 ~ 47,000 and 120,000 ~ 125,000, respectively.

• Food Engineering Progress
• /
• v.21 no.2
• /
• pp.103-109
• /
• 2017

Alginate lyase from Streptomyces violaceoruber was purified by DEAE sephacel chromatography and SP sepharose chromatography. The specific activity of the purified enzyme was 14.6 units/mg protein, representing a 40.6-fold purification of the crude extract. The final preparation thus obtained showed a single band on Tricine-SDS polyacrylamide gel electrophoresis whose molecular weight was determined to be 23.3 kDa. The polyMG block of sodium alginate was hydrolyzed by the purified alginate lyase and then separated by activated carbon column chromatography and bio gel P-2 gel filtration. The main hydrolysates were composed of hetero type M/G-oligosaccharides with the degrees of polymerization (D.P.) being 6 and 8. To investigate the effects of hetero type M/G-oligosaccharides from the sodium alginate on the growth of some intestinal bacteria, cells were cultivated individually on the modified-MRS medium containing D.P. 6 and 8 M/G-oligosaccharides. B. longumgrew 4.25-fold and 6.44-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides compared with those of standard MRS medium. In addition, B. bifidumgrew 3.3-fold and 5.4-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides. In conclusion, D.P. 8 was more effective than D.P. 6 hetero M/G-oligosaccharides as regards the growth of Bifidobacteriumspp. and Lactobacillus spp.

• Microbiology and Biotechnology Letters
• /
• v.44 no.3
• /
• pp.333-340
• /
• 2016

An alkaline protease was purified and characterized from an alkalophilic microorganism, Bacillus sp. DK1122, isolated from soil in central Korea. The optimum temperature and pH for the growth of the producer strain were 40℃ and pH 9.0, respectively. The protease was produced aerobically at 40℃ after 24 h incubation in modified Horikoshi I medium (pH 9.0) containing 0.5% (w/v) glucose, 0.8% (w/v) yeast extract, 0.5% (w/v) polypeptone, 0.1% (w/v) K₂HPO₄, 0.02% (w/v) MgSO₄·7H₂O, 1% (w/v) Na₂CO₃, and 3% (w/v) NaCl. The alkaline protease was purified by 70% ammonium sulfate precipitation of the culture supernatant of Bacillus sp. DK1122, followed by CM-Sepharose chromatography. The molecular weight of the enzyme was estimated to be 27 kDa on the basis of SDS-PAGE. The optimum temperature and pH for the protease activity were 60℃ and pH 9.0, respectively. Addition of CaCl₂ increased the thermal stability of the purified protease, where 90% of protease activity was retained at 60℃ for up to 3 h. Consequently, it is expected that the alkaline protease from this study, exhibiting stability at pH 7–9 and 60℃, may be promising for application in the food and detergent industries.

• Journal of Microbiology
• /
• v.35 no.4
• /
• pp.295-299
• /
• 1997

Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.