• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.315-318
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• 1991

- A bacterial strain No. S-76 which produced pullulanase powerfully was isolated frorn soil. The isolated bacterium was 0.4~$0.6\times 0.8$~1.4 $\mu\textrm{m}$ in size, gram negative, rods, motile and was identified as Aerornonas caviae by Bergey's manual of determinative bacteriology with various characteristics investigated. The highest yield of pullulanase of the strain was obtained by using the following medium: 1% pullulan, soluble starch or corn starch as a carbon sources and 0.5% yeast extract, peptone as nitrogen sources with an initial pH of 9.0. The optimal cutture conditions for production of pullulanase were at $32^{\circ}C$ for 2 days.

• Journal of Life Science
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• v.6 no.2
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• pp.79-86
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• 1996

A bacterium producing pullulanase was from soil, and was identified Bacillus cereus and named as Bacillus cereus JK36. The optimal culture conditions for the efficident production of pullulanase from B. cereus JK36 was obtained by cultivating with the medium composed of 1% pullulan, 1% teast extract, 1% bactopeptone, 0.1% NaH$_{2}$PO$_{4}$, 2H$_{2}$O, 0.02% MgSO$_{4}$\ulcorner7H$_{2}$O at 40$\circ$C, initial pH 6.5 for 70 hours. Using the culture supernatant as crude enzyme, the optimal pH and temperature of the pullulanase of this strain were 6.5 and 50$\circ$C. In effect of pH and temperature on the stability of the enzyme, the enzyme was stable in the range of pH6.0$\sim$9.5 and up to 40$\circ$C, respectively. The hydrolysis product on pullulan was mainly maltotriose.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.73-79
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• 1994

The optimum cultural temperature and time for the pullulanase production by Bacillus cereus subsp. mycoides were 35$\circ $C and 48 hrs, respectively. The addition of egg albumin and casein to the basal medium increased the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. specific activity of the purified enzyme was 82.37 U/mg protein and yield of theenzyume activity was 62.1%. The purified enzuyme showed a single band on ployacrylamide disc gel electrophoresis and its molecular weight was estimated to be 66.,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelcular point for the purified enzyme was pH 5.0. The optimum temperature and pH were 50$\circ $C and pH 6.5, respectively. The purified enzyme was stable below 40$\circ $C and in the pH range of 6.5~10.0 The pullulanase activity was greatly inhited by Ag$^{+}$, Hg$^{2+}$ and EDTA, and its heat stability was increased by the addition of Ca$^{2+}$. The tydrolysis product with the enzyme on pullulan was maltotriose.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.409-416
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• 1992

The optimum cultural temperature and time for the pullulanase production by Bacillus cereus were $15^{\circ}C$ and 72 hrs, respectively. The addition of casein, nutrient broth and egg albumin to the basal medium, respectively, increased greatly the enzyme production. The enzyme was purified by ammonium sulfate fractionation, CM-cellulose and DEAE-cellulose column chromatographies. The specific activity of the purified enzyme was 29.09 U/mg protein and the yield of enzyme activity was 17.1% The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 61,000 by SDSpolyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pH 7.0. The optimum temperature and pH were $40^{\circ}C$ and 6.5. The purified enzyme was stable below $35^{\circ}C$ and in the pH range of 6.5-11.0. It was greatly inhibited by $Ag^{+}$, $Hg^{2+}$ and $Zn^{2+}$, and its thermal stability was increased by the addition of $Ca^{2+}$ Among various substrates, pullulan was favorably hydrolyzed by the purified enzyme and the hydrolysis product 011 pulluIan was maltotriose.

• Journal of Life Science
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• v.7 no.2
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• pp.95-106
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• 1997

The moderately alkalophilic bacterium, identified as Bacillus sp. S-1 , was isolated from soils and effectively secrete extracellular pullulanase. The isolate was moderately alkalophilic since enzyme production occurred at pHs from 6.0 to 10.0. Extracellular crude enzymes of the isolate gave maltotriose as the major product from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes, this isolate secreted extremely high concentration(7.0 units/ml) of pullulanase. The purified pullulanase was moderately alkalophilic and thermoactive; optimal activity was detected at pH 8.0-10.0 and between 50-60$^{\circ}$C. Even at pH 12.0, 10% of S-1 pulluanase activity remained and the strain had broad pH ranges and moderate thermo-stability for their enzyme activities. These results indicate that the new isolate have potential as producer of pullulanase for use in the starch industry.

• The Korean Journal of Food And Nutrition
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• v.9 no.1
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• pp.45-51
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• 1996

Korean traditional Sikhye is made from rice and malt, and It's main product Is maltose. However commercial Sikhye differs from traditional Sikhye because it's main component is sucrose. Sikhye industry faces many problems such as contamination of malt with microorganisms, low amylase activity of malt and technical difficulties. There is no commercial Sikhye which is only using rice and malt by these reasons. To produce the traditional Sikhye free from these problems, it is necessary to restrict the microorganisms of malt and to standardize the amylase activity of malt. In addition, the Introduction of effective control and sanitaric process is required. In Sikhye production. if $\beta$-amylase and isoamylase or pullulanase were added, starch could be saccharified 100% as maltose. Accordingly, this method brings us the low cost of Sikhye.

• KSBB Journal
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• v.22 no.5
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• pp.345-350
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• 2007

Phosphosugars are found in all living organisms and are commercially valuable compounds with possible applications in the development of a wide range of specialty chemicals and medicines. In carbohydrate metabolism, fructose 6-phosphate (F6P) is an essential intermediate formed by phosphorylation of 6' position of fructose in glycolysis, gluconeogenesis, pentose phosphate pathway and Calvin cycle. In glycolysis, F6P lies within the glycolysis metabolic pathway and is produced by isomerisation of glucose 6-phosphate. For large-scale production, F6P could be produced from starch using many enzymes such as pullulanase, starch phosphorylase, isomerase and mutase. In enzymatic reactions carried out at high temperatures, the solubility of starch is increased and microbial contamination is minimized. Thus, thermophile-derived enzymes are preferred over mesophile-derived enzymes for industrial applications using starch. Recently, we reported the production of glucose 1-phosphate (G1P) from starch by Thermus caldophilus GK24 enzymes. Here we report the production of F6P from starch through three steps; from starch to glucose 1-phosphate (glucan phosphorylase, GP), then glucose 6-phosphate (phosphoglucomutase, GM) and then F6P (phosphoglucoisomerase, GI). Using 200 L of 1.2% soluble starch solution in potassium phosphate buffer, 1,253 g of G1P were produced. Then, 30% yields of F6P were attained at the optimum reaction conditions of GM : G1 (1 : 2.3), 63.5$^{\circ}C$, and pH 6.85. The optimum conditions were found by response surface methodology and the theoretical values were confirmed by the experiments. The optimum starch concentrations were 20 g/L under the given conditions.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.