• BMB Reports
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• v.30 no.1
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• pp.46-54
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• 1997

Alkaliphilic Bacillus sp. S-1 secretes a large amount (approximately 80% of total pullulanase activity) of an extracellular pullulanase (PUL-E). The pullulanase exists in two forms: a precursor form (PUL-I: $M_r$ 180,000), and a processed form (PUL-E: $M_r$ 140,000). Two forms were purified to homogeneity and their properties were compared. PUL-I was different in molecular weight, isoelectric point, $NH_2$-terminal amino acid sequence, and stabilities over pH and temperature ranges. The catalytic activities of PUL-I were also distinguishable in the $K_m$ and $V_{max}$ values for various substrates, and in the specific activity for pullulan hydrolysis. PUL-E showed 10-fold higher specific activities than PUL-I. However. PUL-I is immunologically identical to PUL-E, suggesting that PUL-I is initially synthesized and proteolytically processed to the mature form of PUL-E. Processing was inhibited by PMSF, but not by pepstatin, suggesting that some intracellular serine proteases could be responsible for processing of the PUL-I. PUL-I has a different conformational structure for antibody recognition from that of PUL-E. It is also postulated that the translocation of alkaline pullulanase(AP) in the bacterium possibly requires processing of the $NH_2$-terminal region of the AP protein. Processing of the precursor involves a conformational shift. resulting in a mature form. Therefore. precursor processing not only cleaves the signal peptide, but also induces conformational shift. allowing development of active form of the enzyme.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.362-367
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• 1991

The crude enzyme solution obtained by shaking culture of Aeromonas caviae No. S-76 isolated from soil as pullulanase producing bacterium was purified by 50 folds with 21% yield by salting out with ammonium sulfate and column chromatography using DEAE-Sephadex A-50 and Sephadex G-150. The purified pullulanase had a molecular weight of 118, 000 approximately by SDS-polyacrylamide slab gel electrophoresis and pI of 4.3 by isoelectric focusing. And optimum reaction temperature and pH for puHulanase were $50^{\circ}C$ and 8.0, respectively. The purified enzyme was relatively stable at pH 6.0~9.0 and below $45^{\circ}C$. This enzyme synthesized maltosyl-$\beta$-cyclodextrin from mixture of $\beta$-cyclodextrin and maltose.

• 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.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.422-429
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• 1992

The effect of various water-activity depressors, such as pol yo Is, sugars, and polymers, on the conversion yields of the enzymatic synthesis of maltosyl-$\beta$-cyclodextrin from $\beta$-cyc1odextrin and maltose through reverse reaction of pullulanase was investigated. PEG 6000 of concentration of 10% (w/w) was found to be the most acceptable water-activity depressor resulting for increment of conversion yield from 43.0% to 55.9%, corresponding maltosyl-$\beta$-cyc1odextrin concentration of 3.02 g/100 ml H20. Water activity was changed from initial 0.966 to 0.914 upon addition of 20% (w/w) of PEG 6000. The conversion yields were inversely proportional to the water activities, and the increased conversion yield was caused by water activity depression which inhibited the hydrolysis reaction of maltosyl-$\beta$-CD to maltose and $\beta$-cyc1odextrin. The changes of enthalpy ($\Delta$H), entropy ($\Delta$S), and Gibbs free energy ($\Delta$G) were calculated to be 36.788 kJ/mole, 0.067 kJ/mole K. and 14.433 kJ/mole, respectively. The synthesis of maltosyl-$\beta$-CD could be increased substantially by the intermittent feeding of $\beta$-cyclodextrin. PEG 6000 could be separated effectively from reaction mixture using ultrafiltration membrane for reutilization.

• 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.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.444-449
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• 1991

Synthesis of maltosyl-$\beta$-cyclodextrin using maltose ($G_2$) and $\beta$-cyclodextrin ($\beta$-CD) as substrates through the reverse reaction of pullulanase was investigated. The optimal conditions for the condensation reaction were as below: mixing ratio of maltose to $\beta$-CD of 12.7, mixed substrate concentration of 70% (w/w, 70 g/100 ml $H_2O$), and amount of pullulanse of 350 units/100 ml. The concentration of synthesized maltosyl-P-CD concentration was reached up to 2.31 g/100 rnl at above reaction conditions, which corresponded the conversion yield of 43% (w/w, g of branched-CD/g of CD). The synthesis of maltosyl-$\alpha >\gamma >\beta$-CD was also attempted, and conversion yield was in the order of a>y>J3-CDs. Condensation reaction between various maltooligosaccharides ($G-1\sim G_6$ showed that maltose was the most effective oligorner for condensation reaction with $\beta$-CD. To increase the conversion yield various alcohols were added into the reaction mixture, amyl alcohol was found to be the most acceptable alcohol for increasement of convesion yield which increased from 43.0 to 83.0% upon addition of same volume of amyl alcohol into the reaction mixture.

• Korean Journal of Food Science and Technology
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• v.24 no.6
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• pp.568-573
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• 1992

Molecular structural properties of Japonica and Tongil type waxy rice starches were compared. The general properties including water-binding capacity, swelling power at $80^{\circ}C$, intrinsic viscosity, leach loss at $98^{\circ}C$ and ${\beta}-amylolysis$ limit were similar but the chain length was slightly different between the two starches. Similar elution profiles on Sephadex G-50 were obtained when the starches were treated with ${\beta}-amylase$. The pullulanase treated starches however showed different elution patterns each other. The ${\beta}-amylolysis$ of acid-treated (2 days) starches linearly increased as hydrolysis time prolonged. When the acid-treated starches were hydrolyzed with ${\beta}-amylase$ or pullulanase, the elution profiles on Sepharose CL-2B were considerably different from those of native starches.

• 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.

• Journal of Life Science
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• v.9 no.1
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• pp.26-34
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• 1999

A thermostable pullulanase has been isolated and purified from Thermus caldophilus GK-24 to a homogeneity by gel-filtration and ion-exchange chromatography. The specific activity of the purified enzyme was 431-fold increase from the crude culture broth with a recovery of 11.4%. The purified enzyme showed $M_{r}$ of 65 kDa on denaturated and natural conditions. The pI of the enzyme was 6.1 and Schiff staining was negative, suggesting that the enzyme is not a glycoprotein. The enzyme was most active at pH 5.5. The activity was maximal at $75^{\cire}C$ and stable up to $95^{\cire}C$ for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at $4^{\cire}C$ for 24hr. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was simulated by $Mn^{2+}$ ion, }$Ni^{2+}$, $Ca^{2+}$, $Co^{2+}$ ions. The enzyme hydrolyzed the ${\alpha}$-1,6-linkages of amylopectin, glycogens, ${\alpha}$, ${\beta}$-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose and the activity was inhibited by $\alpha$, $\beta$, or $\gamma$-cyclodextrins. The $NH_{2}$-terminal amino acid sequence [(Ala-Pro-Gln-(Asp of Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] was compared with known sequences of various sources and that was compared with known sequences of various sources and that was different from those of bacterial and plant enzymes, suggesting that the enzymes are structurally different.

• Korean Journal of Food Science and Technology
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• v.19 no.4
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• pp.371-376
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• 1987

The structral properties of rice starch and its components were investigated. The intrinsic viscosities of rice starch, amylose and amylopectin solutions were 1.053, 1.772 and 1.596 ml/g, respectively. ${\beta}-amlolysis$ limits of rice starch was 73% and those of amylose and amylopectin were 106 and 56%, respectively. The outer chain length $({\overline{OCL}})$ and inner chain length $({\overline{ICL}})$ of amylopectin were 1.56 and 7.7, respectivety. Debranching of rice starch with pullulanase yield two peaks on Sephadex G-75. The peak I at void volume was principally amylose components and peak II at ${\overline{dp}}$ 25-10, constituted of branched amylopectin. However, the elution profile of debranched amylopectin by pullulanase revealed a bimodal peak distribution at ${\overline{dp}}$ 35-25 hydrolyers and ${\overline{dp}}$ 25-10, and no distinct peak at void volume. The elution profile of the debranched, ${\beta}-amylase$ hydrolyrates of starch and amylopectin yielded major peak at ${\overline{dp}}$ 2-10.