• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.270-276
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• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Journal of Food Hygiene and Safety
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• v.35 no.1
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• pp.83-92
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• 2020

This study investigated the optimal conditions of enzymatic reaction for production of isomaltooligosaccharides (IMO) using rice flour. To manufacture IMO, commercial enzymes (Termamyl 2X, Maltogenase L, Promozyme D2, Fungamyl 800L and Transglucosidase L) were used. The sugar composition and amount of IMO were examined by HPLC with charged aerosol detector (HPLC-CAD) in each manufacturing process. Liquefaction reaction was performed according to different Termamyl 2X concentrations (0.025%, 0.05%, 0.075%, 0.1%) and reaction times (1 h, 2 h). As a result, the reducing sugar content was the highest at 138.26 g/L when 0.075% Termamyl 2X was added for 2 hours. In order to optimize simultaneous saccharification and transglucosylation, experiments on enzyme selection, enzyme concentration and enzyme reaction time were conducted. Reaction with 0.0015% Maltogenase L, 0.05-0.1% Promozyme D2 and 0.1% Tansglucosidase L was effective in decreasing glucose content and increasing content of IMO with a high degree of polymerization. A change in sugar content was observed every 6 hours to determine the optimal reaction time, and the highest IMO was produced after 36 hours of reaction (75.36 g/L). The IMO prepared under optimal conditions showed isomaltose, 35.11 g/L; panose, 11.97 g/L; isomaltotriose, 19.95 g/L; isomaltotetraose, 7.46 g/L; isomaltopentaose, 1.05 g/L at 18 brix and the ratio of IMO in the total sugar was 56.37%.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1060-1069
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• 2013

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at $85^{\circ}C$. The melting temperature of TcCD was determined to be $93^{\circ}C$ by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed ${\alpha}$-cyclodextrin (${\alpha}$-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one ${\alpha}$-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-${\alpha}$-CD, G1-${\beta}$-CD, and G2-${\beta}$-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and $6^2$-${\alpha}$-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

• KSBB Journal
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• v.14 no.6
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• pp.713-717
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• 1999

We have isolated a dextranase and amylase constitutive and hyper-producing mutant, Lipomyces starkeyi JLC26, from Lipomyces starkeyi ATCC74054 after mutation using UV irradiation. After partial purification of dextranase and amylase (together DXAMase;both activities were always co-purified) by ammonium sulfate precipitation, CM-Sepharose column chromatography, the specific activities of amylase and dextranase were 5367 and 3045 unit/mg, respectively. The pH effects for activity and stabiligy of both enzymes were similar to each other: Optimum pH and temperature for activity sere at 5.5 and 37$^{\circ}C$ and optimum ranges for stability were at pH 2.5-5.5 and 4-55$^{\circ}C$, respectively. The reaction end products of dextranase and amylase activities were found to the typical for those of endo-dextranase and endo-amylase. When the carbohydrase and maltotriose were reacted, glucose, maltose, isomaltose, maltotriose, panose and ${\alpha}(1{\rightarrow}6)$glucosylmaltotriose were produced by disproportionation reaction.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.363-369
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• 2016

A putative cyclomaltodextrinase gene (licd) was found from the genome of Listeria innocua ATCC 33090. The licd gene is located in the gene cluster involved in maltose/maltodextrin utilization, which consists of various genes encoding maltose phosphorylase and sugar ABC transporters. The structural gene encodes 591 amino acids with a predicted molecular mass of 68.6 kDa, which shares less than 58% of amino acid sequence identity with other known CDase family enzymes. The licd gene was cloned, and the dimeric enzyme with C-terminal six-histidines was successfully produced and purified from recombinant Escherichia coli. The enzyme showed the highest activity at pH 7.0 and 37℃. licd could hydrolyze β-cyclodextrin, starch, and maltotriose to mainly maltose, and it cleaved pullulan to panose. It could also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. In particular, it showed significantly higher activity towards β-cyclodextrin and maltotriose than towards starch and acarbose. licd also showed transglycosylation activity, producing α-(1,6)- and/or α-(1,3)-linked transfer products from the acarbose donor and α-methyl glucopyranoside acceptor.

• Korean Journal of Microbiology
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• v.53 no.3
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• pp.208-215
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• 2017

A cyclomaltodextrinase (SPCD) gene was cloned from Streptococcus pyogenes ATCC 700294. Its open reading frame consists of 567 amino acids (66.8 kDa), which shows less than 37% of amino acid sequence identity with the other CDase-family enzymes. The homo-dimeric SPCD with C-terminal six-histidines was expressed and purified from Escherichia coli. It showed the highest activity at pH 7.5 and $45^{\circ}C$, respectively. SPCD has the broad substrate specificities against ${\beta}$-cyclodextrin, starch, and maltotriose to produce mainly maltose, whereas it hydrolyzes pullulan to panose. It can also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. Interestingly, it showed much higher activity on ${\beta}$-cyclodextrin and acarbose than that on starch, pullulan, or maltotriose, which makes SPCD distinguished from common CDase-family enzymes. Although SPCD has significantly high acarbose-hydrolyzing activity, it showed negligible transglycosylation activity.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.110-117
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• 1998

A packed-bed reactor with immobilized transglucosidase (TG) was operated to test the possibility of continuous production of isomaltooligosaccharides (IMO) and the effect of concentration and feed rate of substrate solution on the production pattern as well as operational stability The pattern of formation of IMO was the same to the one of soluble TG. The concentrations of glucose and isomaltose produced by the packed-bed reactor were gradually decreased as the flow rates were increased regardless of the concentrations and kinds of maltose solution as substrate. Isomaltotriose showed the same tendency except 10% maltose solution. But the concentration of panose was increased and then decreased as the flow rates were increased. The maximum yield of IMO was 52.1% when 10% (w/v) solution was fed to the reactor at 2 mL./min feed rate. When each 20% and 30% (w/v) solution was respectively used at $0.5{\sim}1.0\;mL/min$, the maximum yield were $39.0{\sim}38.0%\;and\;12.1{\sim}14.2%$. The maximum yield was 36.3% at $0.5{\sim}1.0\;mL/min$ when a commercial maltose product containing 20% maltose was used. The reactor was stably operated at $55^{\circ}C$. 85% and 65% of initial activity was maintained for 144 hours and 288 hours of operation, respectively. A reactor analysis strongly an immobilized TG system could apply to continuous production of IMO.

• Korean Journal of Food Science and Technology
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• v.29 no.6
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• pp.1213-1221
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• 1997

Effects of the addition of three commercial oligosaccharide syrups into a Korean rice cake (Karedduk) on the textural characteristics and retrogradation of the rice cake were examined during the storage for 5 days at $25^{\circ}C$ and $4^{\circ}C$. Each syrups contained maltose (M75), isomaltose and panose (HL), or maltotetraose (G4) as major sugars. The increment (rates) in gumminess, hardness and chewiness during the storage were significantly reduced by replacing rice flour (up to 10%) with the oligosaccharides. The retardation in the textural changes by the oilgosaccharides was more significant when the rice cake was stored at $25^{\circ}C$ than at $4^{\circ}C$. Among the three types, HL exhibited most effective in retarding the textural changes. Thermograms by a differential scanning calorimeter (DSC) showed that the oligosaccharide increased the onset temperatures and enthalpy for the starch melting, but the recrystallinity measured from the enthalpy ratio before and after the storage was significantly reduced by the presence of the oligosaccharide. Especially with 5% HL, the recrystallinity was significantly low (72.7%) compared to rice cake without HL (88.1%). Therefore, HL had great efficiency in retarding starch retrogradation as well as textural changes of the rice cake during the storage.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.106-111
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• 2003

A novel carbohydrolase, which is a DXAMase, containing both dextranase and amylase equivalent activities, was purified from Lipomyces starkeyi KSM22. The purified DXAMase was also found to hydrolyze cellobiose, gentiobiose, trehalose and melezitose, while disproportionation reactions were exhibited with various di- and tri-saccharides, such as maltose, isomaltose, gentiobiose, kojibiose, sophorose, panose, maltotriose, and isomaltotriose with various kinds of oligosaccharides produced as acceptor reaction products. Furthermore, the purified DXAMase hydrolyzed raw waxy rice Starch and produced maltodextrin to the extent of 50% as a glucose equivalent.

• BMB Reports
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• v.34 no.4
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• pp.347-354
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• 2001

$\alpha$-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and $90^{\circ}C$, and was stable from pH 6.0 to 85 and up to $90^{\circ}C$. The enzyme had a half-life of 85 minutes at $90^{\circ}C$. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of $\alpha$-1,6-glucosidic linkages of isomaltosaccharides and panose, $\alpha$-1,3-glycosidic bond of nigerose and turanose, and $\alpha$-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the $\alpha$-amylase family. We suggest that this monomeric, thermostable, and broad-acting $\alpha$-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel $\alpha$-glucosidase.