• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.957-963
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• 2001

Bacillus subtilis YL-1004 was isolated from soil for the development of agents to control dental caries. This strain produced an extracellular lytic enzyme that hydrolyzed the Streptococcus mutans cell wall. The lytic enzyme was purified to homogeneity by affinity chromatography and gel permeation chromatography to give a single band on SDS-PAGE and non-denaturing polyacrylamide gel electrophoresis. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography to be 38 kDa and the PI to be 4.3 from isoelectric focusing. Sirty $\%$ of its lytic activity remained after incubation at $50^{\circ}C$ for 30 min, and its optimal temperature was $37^{\circ}C$ . The enzyme showed its highest activity at pH 8.0 and was stable at pHs ranging from 4.0 to 9.0. Treatment with several modifiers showed that a cysteine residue was involved in the active site of the enzyme. This lytic enzyme from Bacillus subtilis YL-1004 exhibited specificity towards Streptococci and also showed autolytic activity on Bacillus subtilis YL-1004.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.467-471
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• 1986

Yeast cell wall lytic enzyme was purified from Fusarium moniliforme by ammonium sulfate fractionation and gel column chromatography. The lytic activity was found to consist of three enzyme activities which were resolved on Sephadex G-100. The first peak on chromatogram exhibited proteolytic, lytic and laminarinase activities, and the second had both lytic and laminarinase activities, whereas the third peak was shown to contain lytic activity only. Three enzyme activities showed the synergistic effect and reducing agents accelerated the yeast roil wall lysis. This indicates that lytic, proteolytic and laminarinase activity acted cooperatively in the lysis of intact cells. Tannic acid precipitate of crude enzyme constituted of three enzyme activities had a high lytic activity on viable yeast cell and has proved useful in yeast protoplast formation.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.573-579
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• 1995

A Bacillus strain capable of producing an extracellular malto-oligosaccharides forming $\alpha $-amylase was isolated from soil and designated as Bacillus sp. SUH4-2. The enzyme was purified by ammonium sulfate fractionation, DEAE-Toyopearl and Mono-Q HR 5/5 column chromatographies using a FPLC system. The specific activity of the enzyme was increased by 16.1-fold and the yield was 13.5%. The optimum temperature for the activity of $\alpha $-amylase was 60-65$\circ$C and more than 50% of initial activity was retained after the enzyme was incubated at 60$\circ$C for 40 min. The enzyme was stable over a broad pH range of 5.0-8.0 and the optimum pH was 5.0-6.0. The molecular weight of the enzyme was determined to be about 63.6 kD and isoelectric point was around 5.8. The enzyme activity was strongly inhibited by Mn$^{2+}$, Ni$^{2+}$, and Cu$^{2+}$ ; slightly by Ca$^{2+}$. The purified enzyme produced starch hydrolyzates containing mainly maltose and maltotriose from soluble starch. The starch hydrolyzates were composed of 11% glucose, 59% maltose, 25% maltotriose and 5% maltotetraose.

• Journal of Microbiology
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• v.35 no.2
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• pp.134-140
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• 1997

Botrytis cinerea T91-1 has been shown to produce at least four different polygalacturonases into a liquid medium containing citrus pectin, a carbon sousrce. One of the enzymes, which had an apparent molecular weight of 66 kDa estimated by denatured polyacrylamide gel electrophoresis, was purified to electrophoretic homogeneity by a series of procedures including a cetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. The molecular weight of native enzyme was determined to be 64 kDa by gel permeation chromatography indicating the enzyme to be a single polypeptide chain. By viscometric analysis, the enzyme was revealed as exo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Mg^{2+}$, 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 5$0^{\circ}C$. And the enzyme showed optimal pH values between 4.0 and 5.0. The enzyme was stable upto 12 hours in the range of pH 3 to 8 and at temperature below 3$0^{\circ}C$.

• BMB Reports
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• v.29 no.4
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• pp.335-342
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• 1996

Porcine liver cysteinesulfinic acid decarboxylase was purified approximately 460-fold by means of ammonium sulfate fractionation and sequential column chromatographic separation with Sephadex G-100, DEAE-cellulose and hydroxylapatite. The enzyme has a flat pH profile with maximum activity occurring between pH 6.0 and 7.6. Pyridoxal 5'-phosphate must be present in all buffers used for purification procedures in order to stabilize the enzyme. Addition of sulfhydryl reagents such as 2-mercaptoethanol are also necessary to maintain maximum enzyme activity throughout purification. The absorption spectrum shows that cysteinesulfinic acid decarboxylase is a pyridoxal 5' -phosphate-containing protein. The major absorption is at 280 nm with two smaller absorption regions, one at 425 nm which is ascribed to a Schiffs base between pyridoxal phosphate and protein, and another at 325 nm which is thought to be due to the interaction of 2-mercaptoethanol with the Schiffs base. A number of divalent cations tested did not affect enzyme activity with the exception of mercury, copper, and zinc which are inhibitory. The partially purified enzyme has an apparent $K_m$ of 0.94 mM for cysteinesulfinate. Cysteic acid is a competitive inhibitor of the enzyme with a $K_i$ of 1.32 mM. The molecular weight of the enzyme was estimated to be about 79,600 by using Sephadex G-200 column chromatography.

• Preventive Nutrition and Food Science
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• v.2 no.1
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• pp.71-76
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• 1997

Pullulanase was produced from the Klebisella pneumonias NFB_320 with the conmposition of 0.1% pullualn 1.5% yeast extract, 0.2% $K_2$HPO$_4$ and 0.02% MgSO$_4$.7$H_2O$(pH5.5). The optimum temperature for activity of the pulluanase was 3$0^{\circ}C$ and the highest yield of the enzyme was obtained after cell growth at 3$0^{\circ}C$ for 18hr, and maintained until 24hr cultivation. The pullulanase was successively purified 52.6 folds with 7.8% yield by acetone precipitation. DEAE-cellulose column chromatography and gel fitrations. The purified enzyme hydrolyzed pullulan into maltotriose exclusively. Chemical and physical properties of purified pullulanase from Klebisella pneumonias NFB-320 were examined. The optimum pH and temperature for enzyme activity were 5.0 and 6$0^{\circ}C$, respectively. The enzyme was stable between pH4 and 7, and up 5$0^{\circ}C$. The effect of mo-dification on the rate of enzyme reaction was studies with various chemicals and metal ions. The enzyme has been found to be inactivated by I$_2$ and N-bromosussinimide(NBS), which probably indicated the involve- ment of tryptophan residues in the active center of the enzyme.

• Korean Journal of Food Science and Technology
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• v.18 no.4
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• pp.288-293
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• 1986

A raw starch-digesting enzyme from Rhizopus oryzae was purified by ammonium sulfate fractionation, DEAE-sephacel column chromatography and Sephadex G-150 gel filtration. The specific activity of purified enzyme was 45.2 Ulmg protein and the yield was 16.2%. The purified enzyme was found to be homogeneous bypolyacrylamide gel electrophoresis and its molecular weight was estimated to be 67,000 by SDS-polyacrylamide gel electrophoresis, and also the enzyme had Km value of 4.082 mg/ml for raw corn starch. The optimal temperature and pH for the enzyme activity were $50^{\circ}C$ and 4.0-5.0, respectively. Reaction product of raw corn starch by purified enzyme was glucose mainly.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.230-234
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• 2003

An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. When the enzyme was chemically modified with PMSF, which specifically reacted with serine residue on the enzyme, the activity was eliminated. The endopeptidase activity was inhibited by the modifier which chemically modified carboxyl group of aspartate and glutamate. PLP, which would modify lysine residue, did not affect the endopepetidase activity to a greater extent. This demonstrates that serine and aspartate (or glutamate) residues of enzyme would participate in a important function of the endopeptidase activity.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.573-578
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• 2005

Pyrimidine nucleotide N-ribosidase (pyrimidine 5'-nucleotide phosphoribo(deoxyribo)hydrolase/pyrimidine 5'-nucleotide nucleosidase, EC 3.2.2.10) catalyzes the breakdown of pyrimidine 5'-nucleotide into pyrimidine base and ribose(deoxyribo)-5-phosphate. However, detailed characteristics of the enzyme have not yet been reported. The enzyme was purified to homogeneity 327.9-fold with an overall yield of $6.1\%$ from Pseudomonas oleovorans ATCC 8062. The enzyme catalyzed cytidine monophosphate (CMP) and uridine monophosphate (UMP), but not adenosine monophosphate (AMP) and guanosine monophosphate (GMP). The enzyme optimally metabolized CMP at pH 6.0 and UMP at around 8.5, and the optimum temperature for the overall enzyme reaction was found to be $37^{\circ}C$. The $K_m$ values of the enzyme for CMP (at pH 6.0) and UMP (at pH 8.5) were 1.6 mM and 1.1 mM, respectively. AMP, deoxyCMP, and deoxyUMP were very effective inhibitors of the reaction. Double-reciprocal plots obtained in the absence and in the presence of AMP revealed that this inhibitory effect was of the mixed competitive type with respect to the breakdown of CMP and of the noncompetitive type with respect to the breakdown of UMP. In the presence of AMP, the enzyme followed sigmoid kinetics with respect to each substrate.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.1
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• pp.32-37
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• 2006

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria, Ochrobactrum anthropi SY509, which was isolated from soil samples. O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to $70^{\circ}C$. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membranebound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.