• 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.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.

• 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.

• KSBB Journal
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• v.9 no.5
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• pp.499-505
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• 1994

Bacillus macerans cyclodextrin glucanotransferase(EC 2.4.1.19: 1, 4-${\alpha}$-D(1, 4-${\alpha}$-glucano)-transferase, CGTase) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography. The molecular weight of the enzyme was 67,000, consisting of a subunit. The enzyme converted starch into ${\alpha}$-, ${\beta}$-, and ${\gamma}$-CD in the relative amounts of 1:1.68:0.32, respectively. In the early reaction period, maltohexose was formed mainly by the coupling reaction of ${\alpha}$-CD with D-glucose and then other oligosaccharides. Maltotetrose was formed mainly from ${\alpha}$-CD in the initial stage of hydrolysis of the enzyme and then small amount of other oligosaccharides. Maltotriose was a good substrate for the enzyme and maltosyl or D-glucopyranosyl group can be transfered from this sugar. In this work, D-glutosyl transfer was premiered.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.792-799
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• 2007

A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae(abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at $85^{\circ}C$. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and $\alpha$-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotriose, and $6-O-\alpha-maltosyl-\beta-cyclodextrin(G2-\beta-CD)$ to maltose and $\beta$-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to $G2-\beta-CD$. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.