• Food Science and Biotechnology
• /
• v.16 no.6
• /
• pp.902-909
• /
• 2007

Granular maize starch was treated with Thermus scotoductus 4-${\alpha}$-glucanotransferase (${\alpha}$-GTase), and its physicochemical properties were determined. The gelatinization and pasting temperatures of ${\alpha}$-GTase-modified starch were decreased by higher enzyme concentrations. ${\alpha}$-GTase treatment lowered the peak, setback, and [mal viscosity of the starch. At a higher level of enzyme treatment, the melting peak of the amylose-lipid complex was undetectable on the DSC thermogram. Also, ${\alpha}$-GTase-modified starch showed a slower retrogradation rate. The enzyme treatment changed the dynamic rheological properties of the starch, leading to decreases in its elastic (G') and viscous (G") moduli. ${\alpha}$-GTase-modified starch showed more liquid-like characteristics, whereas normal maize starch was more elastic and solid-like. Gel permeation chromatography of modified starch showed that amylose was degraded, and a low molecular-weight fraction with $M_w$ of $1.1{\times}10^5$ was produced. Branch chain-length (BCL) distribution of modified starch showed increases in BCL (DP>20), which could result from the glucans degraded from amylose molecules transferred to the branch chains of amylopectin by inter-/intra-molecular transglycosylation of ${\alpha}$-GTase. These new physicochemical functionalities of the modified starch produced by ${\alpha}$-GTase treatment are applicable to starch-based products in various industries.

• KSBB Journal
• /
• v.9 no.5
• /
• pp.499-505
• /
• 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 Life Science
• /
• v.14 no.3
• /
• pp.435-440
• /
• 2004

The gene encoding a extremely thermostable 4-$\alpha$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis, was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence of the enzyme was distantly related to other functionally-related ones, such as D-enzymes. The enzyme is of industrial interest because of a novel activity of producing cycloamylose and is also important for fundamental studies of protein, sugar-metabolizing enzymes. In this paper, the overexpression of 4-$\alpha$-glucanotransferase in E. coli was carried out expression vector system with lac and T7 promoters. The enzyme was successfully overexpressed, and purified by the heat treatment of a cell-free extract, successive Butyl-Toyopearl and Mono Q chromatographies. The purified recombinant enzyme showed the same specific activity and the same mobility in SDS-PAGE as natural enzyme.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.29 no.1
• /
• pp.49-56
• /
• 2000

The 2-O-$\alpha$-D-glucopyranosyl L-ascorbic acid (AA-2G) which was enzymatically glucosylated with the cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] from Bacillus sp. JK-43 has been reported previously. The presnet experiments examined the optimal conditons for the productio of AA-2G from AA and soluble starch, and characterized the properties of the CGTase from Bacillus sp. JK-43. The reaction mixture for the maximal production of AA-2G was followings; 12% total substrate concentration, 1,400 usits/mL of CGTase and a mixing ratio of 2 : 3(g or AA : g of soluble starch). Under this condition, 1.76mM of AA-2G, which corresponded to 2.53% yield based on AA, was produced after incubation for 24hrs at 45$^{\circ}C$ (pH 5.5). The optimum pH and temperature for the CGTase activity were 6.0 and 45$^{\circ}C$, respectively. The enzyme was stable at pH 5.5 to 9.5, and at temperature up to 5$0^{\circ}C$. The thermostability of the enzyme could be enhanced up to 6$0^{\circ}C$ by the addition of 30mM CaCl2.

• Journal of Life Science
• /
• v.21 no.2
• /
• pp.221-226
• /
• 2011

The gene encoding 4-$\alpha$-glucanotransferase (mgtA) from Thermotoga neapolitana was cloned and expressed in Escherichia coli in order to investigate whether this enzyme was capable of producing cycloamylose for industrial applications. MgtA was purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatographies. The size of the enzyme as determined by SDS-PAGE was about 52 kDa, which was in good agreement with its deduced molecular mass of 51.9 kDa. The optimal temperature and pH for the activity of the 4-$\alpha$-glucanotransferase was found to be $85^{\circ}C$ and 6.5, respectively. The enzyme hydrolyzed the 1,4-$\alpha$-glucosidic bonds in oligomeric 1,4-$\alpha$-glucans and transferred oligosaccharides (maltotriose being the shortest one) to acceptor maltodextrins. However, the enzymes had no activity against pullulan, glycogen, and other di- or trioligosaccharides with rare types of $\alpha$-bond. MgtA is distinguished from 4-$\alpha$-glucanotransferase from Thermotoga maritima in that it can convert maltotriose into maltooligosaccharides. The treatment of glucoamylase after the reaction of MgtA with maltotriose, maltotetraose, maltopentaose, or maltohexaose as sole substrate revealed that MgtA yielded linear maltooligosaccharides instead of cycloamylose.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.27 no.4
• /
• pp.609-617
• /
• 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
• /
• v.32 no.1
• /
• pp.29-36
• /
• 2004

$\alpha$-Cyclodextrin glucanotransferase excreted from a newly isolated Geobacillus thermosacchalytycus was purified through the ultrafiltraion, hydrophobic Sepharose CD-4B affinity chromatography, and gel filtration on Sephadex G-75, respectively. The molecular weight of the purified CGTase was 69 kDa and its N-terminal amino acid sequence was determined to be Asn-Leu-Asn-Lys-Val-Asn-Phe-Val-Ser-Asp-Val-Val-Val-Gln-Ile. The optimum pH and temperature were pH 6.0 and$ 60^{\circ}C$, respectively, and stably at the pH range of 6.0-8.0 and $60^{\circ}C$ in the presence of $Ca^{++}$. The excreted CGTase from the thermophilic G. thermosacchalytycus was $\alpha$-type showing a high coupling activity for the transglycosylation on various glucosides. The coupling reaction was carried out according to the random ternary complex mechanism.m.

• KSBB Journal
• /
• v.15 no.6
• /
• pp.556-559
• /
• 2000

Cyclodextrin Glucanotransferase(EC 2.4.1.19 : 1,4-${\alpha}$-glucano) transferase, cyclizing; CGTase) can be separated and recovered in an aqueous two-phase system composed of poly(ethylene glycol)(PEG)/dextran and PEG/salt. In an aqueous two-phase system consisting of PEG 35000 (5%) and dextran T2000 (7%), all cell and debris were collected at the interphase. CGTase partitioned to the denser dextran phase at an yield of 83.4%. On the other hand, in an aqueous two-phase system consisting of PEG 35000 (10%) and sodium phosphate (15%), CGTase partitioned to the denser salt phase at an yield of 95.5%. In order to recover CGTase using an aqueous two-phase system, the PEG/salt system proved to be more efficient than the PEG/dextran system in terms of yield and cost.

• Food Science and Biotechnology
• /
• v.17 no.4
• /
• pp.705-712
• /
• 2008

Sweet potato starch was modified using Thermus aquaticus $\alpha$-1,4-glucanotransferase ($Ta{\alpha}GT$), and its structural and rheological properties were investigated. $Ta{\alpha}GT$-modified starch had a lower amylose level and molecular weight than raw starch. The chain length distribution showed an increased number of short and long branched chains and the formation of cycloamyloses. Compared with raw starch, $Ta{\alpha}GT$-modified starch displayed a lower gelatinization enthalpy and a wider melting temperature range. The X-ray diffraction of $Ta{\alpha}GT$-modified starch was a weak V-type pattern with distinct sharp peaks at 13 and $20^{\circ}$. Scanning electron micrographs of modified starch exhibited big holes on the surface and the loss of granular structure. The frequency sweep measurement revealed that the gel of $Ta{\alpha}GT$-modified starch was more rigid than raw starch gel. However, the structure of modified starch gel was destroyed by heating at $75^{\circ}C$, and a firm gel was re-formed by subsequent storage at $5^{\circ}C$, indicating thermoreversible property.

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