• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.568-574
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• 1998

Two types of chitosanases produced from Aspergillus fumigatus KH-94 were purified by ion exchange and gel permeation chromatography. Molecular weights of the enzymes are 22.5 kDa (chitosanase I) and 108 kDa (chitosanase II). pI, optimum pH, and temperature of chitosanase I are 7.3, 5.5, and 70-$80^{\circ}C$, respectively, and those of chitosanase II are 4.8, 4.5~5.5, and 50~$60^{\circ}C$, respectively. Activities of both chitosanases were increased by $Mn^{2+}$ but inhibited by $Cu^{2+}$ and $Hg^{2+}$ . Chitosanase I has endo-splitting activity that hydrolyzes chitopentaose, chitohexaose, and chitosan to chitobiose, chitotriose, and chitotetraose, whereas chitosanase II has exo-splitting activity that hydrolyzes chitobiose and chitosan to glucosamine. Chitosanase II was found to have transglycosylation activity also in the reaction of 2% more chitooligosaccharides as a substrate and at the initial reaction. The higher degree of deacetylation, the stronger activities of chitosanase Iand II toward chitosans. Both chitosanases could hydrolyze chitosan and glycol chitosan but not chitin, cellulose, and carboxymethyl cellulose. To produce higher degree of polymerization of chitooligosaccharides, chitosanase I was used and yielded 80% of recovery.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.1-8
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• 1999

Most carbohydrates exist in nature in an insoluble state, which reduces their susceptibility towards various carbohydrases. Accordingly, they require intensive pretreatment for structural modification to enhance an enzyme reaction. The direct conversion of insoluble carbohydrates has distinct advantages for special types of reaction, especially exo-type carbohydrase; however, its application is limited due to structural constraints. This paper introduces two novel heterogeneous enzyme reaction systems for direct conversion of insoluble carbohydrates; one is an attrition coupled enzyme reaction system containing attrition-milling media for enhancing the enzyme reaction, and the other is a heterogeneous enzyme reaction system using extruded starch as an insoluble substrate. The direct conversion of typically insoluble carbohydrates, including cellulose, starch, and chitin with their corresponding carbohydrases, including cellulase, amylase, chitinase, and cyclodextrin glucanotransferase, was carried out using two proposed enzyme reaction systems. The conceptual features of the systems, their reaction characteristics and mechanism, and the industrial applications of the various carbohydrates are analyzed in this review.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1758-1764
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• 2007

Synthesis of oligosaccharides during milk fermentation was attempted by inoculating Leuconostoc citreum with Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus, and Streptococcus thermophilus as starters. Dextransucrase of Ln. citreum worked as a catalyst for the transglycosylation reaction of sugars; sucrose was added as the glucose donor, and lactose or maltose acted as the acceptor compound for the reaction. When 4% sucrose was added in milk, glucosyl-lactose was synthesized (about 1%, w/v) after 1-2 days of fermentation at 15 or $25^{\circ}C$. Alternatively, when sucrose and maltose (2% each, w/v) were added, panose (about 1 %, w/v) and other isomaltooligosaccharides were made in a day at $15-35^{\circ}C$. Growth patterns of lactobacilli and streptococci starters were not affected by the coculture of leuconostoc starter, but the rate of acid synthesis was slightly slowed at every temperature. Addition of sugars in milk did not give any adverse effect on the lactate fermentation. Accordingly, the use of leuconostoc starter and addition of sugars in milk allowed the production of oligosaccharides-containing fermented milk, and application of this method will facilitate the extensive development of synbiotic lactate foods.

• Korean Journal of Microbiology
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• v.27 no.1
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• pp.35-42
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• 1989

The mode of transglycosylation reaction observed during the action of low-molecular-weigh $\beta$-D-glucosidase ($\beta$-D-glucoside glucohydrolase, EC3.2.1.21) purified from Trichoderma koningii ATCC 26113 was investigated using $^{1}H$-NMR spectroscopy. The enzyme was purified by the series of procedures including ammonium sulfate precipitation, and fractionations by column chromatographies on Bio-Gel P-150, DEAE-Sephadex A-50, and SP-Sephadex C-50. The final purification was performed by the band eluation after preparative polyacrylamide gel electrophoresis. The enzyme showed its molecular size of 78,000 through the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point of 5.80 through the analysis of analytical isoelectric focusing. The H-1 proton resonances were analyzed. After the reaction of the enzyme with cellobiose, the reaction products were separated by high performance liquid chromatography using refractive index detector. H-1 resonances of the products were consisted with those of gentiobiose [$\beta$-D-glucopyranosyl--(1,6)-D-glucopyranose], and cellotriose [$\beta$-D glucopyranosyl-(1,4)-$\beta$-D-glucopyranosyl]-(1,4)-D-glucopyranose] with minor resonances of sophorose [$\beta$-D-glucopyranosyl-(1,2)-D-glucopyranose], respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.4
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• pp.609-617
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• 1998

A microorganism capable of producing high level of extracellular cyclodextrin glucanotransferase(EC 2.4.1.19 ; CGTase) was isolated from Kimchi. 2-O-$\alpha$-D-glucopyranosyl L-ascorbic acid(AA-2G) was synthesized by transglycosylation reaction of CGTase using starch as a donor and L-ascorbic acid as an acceptor. The isolated strain S-6 was identified as Bacillus sp. S-6. The maximal CGTase production was observed in a medium containing 0.5% soluble starch, 1% yeast extract, 1% NaCO3, 0.1% K2HPO4, and 0.02% MgSO4 with initial pH 8.0. The strain was cultured at 37$^{\circ}C$ for 40 hr with reciprocal shaking. Using the culture supernatant as crude enzyme, the optimal pH and temperature of the CGTase activity of this strain were 7.0 and 4$0^{\circ}C$. In the effects of pH and temperature on the stability of the enzyme, the enzyme was stable in the range of pH 6.0~10.0 and up to 45$^{\circ}C$, respectively.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.218-227
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• 2022

Glycosylation of aesculetin was performed using amylosucrase from the hyperthermophilic bacterium Deinococcus geothermalis DSM 11300 to improve the solubility and biological activity of aesculetin. A newly synthesized aesculetin glycoside was identified as α-cichoriin (aesculetin 7-α-D-glucoside) by nuclear magnetic resonance analysis. The solubility of α-cichoriin was 11 times higher than that of aesculetin because of the attached glucose moiety. Aesculetin and α-cichoriin had no significant effect on the proliferation of normal cells, such as RAW 264.7, but they showed a cell proliferation inhibitory effect on B16F10 melanoma cells. Unlike treatment with aesculetin and α-cichoriin, aesculin (aesculetin 6-β-D-glucoside) showed no antiproliferative activity in B16F10 cells. Based on the molecular structures of aesculin and α-cichoriin, the position where glucose binds to aesculetin and the anomeric configuration between glucose and aesculetin are thought to be important for exerting an antiproliferative effect on the B16F10 cell line. Based on these results, we propose that α-cichoriin, the α-glycosylated form of aesculetin, may serve as a model for developing phytochemical analogs with therapeutic potential for the treatment of diseases associated with tumor cell proliferation without cytotoxicity to normal cells.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.370-378
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• 1989

An Alkalophilic Bacillus circulans that can produce significant amount of cyclodextrin glucanotransferase (CGTase) was newly isolated from soil. The culture filtrate was successively purified by ($NH_4$)$_2$$SO_4$precipitation, DEAE-Sephadex column chromatography, and Sephadex G-100 column chromatography. The enzymatic properties, including molecular weight, optimal pH and temperature, stability, and kinetic parameters, were determined. The cyclodextrin synthesis reaction catalized by the purified CGTase was also studied. The sweet potato and corn starch were found to be the most suitable substrates with 60% conversion to cyclodextrin. The highest conversion was achieved at the CGTase concentration of 900-1,100 units/g of soluble starch. The purified CGTase could also catalize the transglycosylation on stevioside.

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.829-835
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• 2009

A dextransucrase (LcDS) gene from Leuconostoc citreum HJ-P4 has been amplified and cloned in E. coli. The LcDS gene consists of 4,431 nucleotides encoding 1,477 amino acid residues sharing 63-98% of amino acid sequence identities with other known dextransucrases from Leuc. mesenteroides. Interestingly, 0.1 mM of IPTG induction at $15^{\circ}C$ remarkably increased the LcDS productivity to 19,187 U/I culture broth, which was over 330-fold higher than that induced at $37^{\circ}C$. Optimal reaction temperature and pH of LcDS were determined as $35^{\circ}C$ and pH 5.5 in 20 mM sodium acetate buffer, respectively. Meanwhile, 0.1 mM $CaCl_2$ increased its activity to the maximum of 686 U/mg, which was 2.1-fold higher than that in the absence of calcium ion. Similar to the native Leuconostoc dextransucrase, recombinant LcDS could successfully produce a series of isomaltooligosaccharides from sucrose and maltose, on the basis of its transglycosylation activity.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.442-449
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• 1998

Several transglucosylated xylitols were synthesized using intermolecular transglucosylation reaction of cyclodextrin glucanotransferase (CGTase) and their bifidogenic effects were investigated. The CGTase from Thermoanaerobacter sp. showed the highest transglycosylation activity on xylitol compared to those obtained from other strains. Extruded starch was identified to be the most suitable glucosyl donor for transglucosylation reaction on xylitol molecule by CGTase. The optimum reaction conditions for transglucosylation were also studied using extruded starch as a glucosyl donor. The transglucosylated xylitols were purified by activated carbon column chromatography with ethanol gradient elution from 0 to 18%, and their chemical structures were analyzed by fast atom bombardment mass spectrometer, $\^$13/C-nuclear magnetic resonance spectrometer, and enzyme digestion method. Two transglucosylated xylitol, F-I and F-II, which had one or two glucose molecules attached to maternal xylitol by ${\alpha}$-1,4-linkage, were mainly obtained. F-II showed increased stimulation effect on the growth of Bifidobacterium breve compared to xylitol, indicating the possibility utilized as a new functional alternative sweetners having bifidogenic effects.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1809-1813
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• 2006

A gene corresponding to 4-${\alpha}$-glucanotransferase (${\alpha}GTase$) was cloned from the thermophilic bacterium Thermus brockianus. The nucleotide sequence analysis showed that the ${\alpha}GTase$ gene is composed of 1,503 nucleotides and encodes a polypeptide that is 500 amino acids long with a calculated molecular mass of 57,221 Da. The deduced amino acid sequences of Thermus brockianus ${\alpha}GTase$ (TBGT) exhibited a high level of similarity to the amino acid sequence of ${\alpha}GTase$ of Thermus thermophilus (86%), but low level of homology to that of E. coli (26%). The TBGT gene was overexpressed in E. coli BL21, and the corresponding recombinant enzyme was efficiently purified by Ni-NTA affinity chromatography. The enzymatic characteristics revealed that optimal pH and temperature were pH 6 and $70^{\circ}C$, respectively. Most interestingly, TBGT reacted with small oligosaccharides, especially maltotriose, to form various maltooligosaccharides by using its disproportionation activity.