Analysis of the Gene Encoding Cyclomaltodextrinase from AlkalophilicBacillussp. I-5 and Characterization of Enzymatic Properties

Kim, Tae Jip; Shin, Jong Heon; Oh, Jung Hwa; Kim, Myo Jeong; Lee, Soo Bok; Ryu, Sangryeol; Kwon, Kisung; Kim, Jung Wan; Choi, Eon Ho; Robyt, John F.; Park, Kwan Hwa

doi:10.1006/abbi.1998.0639

Cited by 52 publications

(42 citation statements)

References 28 publications

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“…2). A search with the BLAST program (43) revealed that the protein encoded by the ORF is homologous to neopullulanases (19,26), CDases (22,35), maltogenic amylase (6), and other amylolytic enzymes. It was suggested that the ORF encoded a kind of amylolytic enzyme.…”

Section: Resultsmentioning

confidence: 99%

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Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

et al. 2001

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A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) ␣-cyclomaltodextrin (␣-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991Microbiol. 65: -1997Microbiol. 65: , 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95°C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed ␤-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to ␣-CD. However, the CBP exhibited higher thermostability in the presence of ␣-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.Cyclomaltodextrins (CDs) are cyclic oligosaccharides consisting of ␣-1,4-linked 6-, 7-, or 8-glucopyranose units, usually referred to as ␣-, ␤-, or ␥-CDs, respectively. CDs possess a unique torus shape and the polar hydroxyl groups are oriented toward the outside, keeping the interior cavity relatively hydrophobic. Therefore, CDs are soluble in water and the hydrophobic environment of the cavity enables them to form inclusion complexes with many organic and inorganic molecules, thereby changing the physical and chemical properties of the included compounds. This is the basis of broad applications in the food, cosmetic, and pharmaceutical industries (2, 28).CDs are formed enzymatically from starch by the action of cyclomaltodextrin glucanotransferase ( In K. oxytoca M5a1, the novel starch degradation pathway via CD, including the extracellular conversion of starch into CDs by CGTase and uptake of the CDs by a specific system followed by intracellular linearization by a CDase, was proposed. In addition, the genes responsible for starch metabolism were clustered on the chromosome. Until now, no other organisms possessing both CGTase and CDase for the synthesis and degradation of CDs have been found.Recently, we have isolated a hyperthermophile, Thermococcus sp. strain B1001, which produces a unique CGTase that can catalyze predominantly the formation of ␣-CD (Ͼ8...

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Section: Resultsmentioning

confidence: 99%

“…(3), B. sphaericus strain ATCC 7055 (15), Klebsiella oxytoca strain M5a1 (11,12), and alkalophilic Bacillus sp. strain I-5 (22) were studied.…”

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Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

et al. 2001

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“…1), showed increased thermostability and catalytic activity in the presence of the Ca 2 ion. 40) Conclusively, the Ca2 site can be divided into two groups depending on the contribution to the thermostabilization of the enzyme; one (type I) is the group having Asx Asx Asn Asx Gly Asp as TVAII, CDI 5 and CGT251 (Asx is Asp or Asn); the other (type II) is the group having Asn Asx Ile Ser Gly Asp as BSNPL and ThMA (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

Kim

et al. 2005

J. Appl. Glycosci.

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Thermus maltogenic amylase (ThMA), one of the cyclodextrin (CD)-degrading enzymes, is expected to have a calcium-binding site (Ca2 site) based on multiple sequence alignments. In spite of the presumption of the Ca2 site in ThMA, however, its thermostability is independent of calcium ions. In order to investigate the effect of calcium ions on thermostability, two mutations (Ile152Asn and Ser153Asn) were introduced into Ca2 sites of a thermostabilized ThMA mutant (ThMA-DM2) using site-directed mutagenesis. The resultant mutant (ThMA-DM2-Ca) showed highly improved thermostability in the presence of calcium ions. The relative hydrolysis activity of ThMA-DM2-Ca also increased in comparison with that of ThMA-DM2 whereas transglycosylation activity was not affected by substitutions. The result confirmed that the residues located in the Ca2 site (Ile152 and Ser153) played critical role in stabilizing CD-degrading enzymes.

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“…Several CDases have a transglycosylation activity as well as hydrolysis activity. 20,39) These enzymes transfer a glucose and a maltose moiety from the substrates to an acceptor. There have been no other reports of a CDase which is capable of transferring maltotriose and thus, this transglycosylation profile of Cda is a novel feature in CDases.…”

Section: Analysis Of Reaction Product Compositionmentioning

confidence: 99%

“…A2 5a, 19) Bacillus sp. I 5, 20) Clostridium thermohydrosulfuricum 39E, 21) Flavobacterium sp. 92, 22) Klebsiella oxytoca M5a1, 23) Paenibacillus sp.…”

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Characterization of Two γ-Cyclodextrin-specific Enzymes from <i>Bacillus clarkii</i> 7364

et al. 2010

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Cyclodextrin (CD) has a closed ring structure consisting of D glucose residues linked through α (1,4) linkages. The most common CDs consist of six, seven, or eight glucose residues, and are named α , β and γ CD, respectively. CDs have a hydrophilic outer surface and a relatively hydrophobic internal cavity. CDs are able to form complexes with various molecules. The formation of complexes leads to changes in the chemical and physical properties of the molecule including stability and solubility; thus there have been extensive applications of CDs in the food, cosmetic, agricultural and pharmaceutical industries. 1) Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) forms CDs from starch and related α (1,4) linked glucose polymers through intramolecular transglycosylation. 2) This enzyme belongs to glycoside hydrolase family 13 (α amylase family), based on its amino acid sequence. 3 5) Members of this family show various reaction specificities but have the following common features: a (β α)8 barrel structure and four highly conserved regions, regions I IV, in the primary sequence. The conserved regions include invariant catalytic residues, two Asp residues and one Glu residue, and other amino acids involved in transition state stabilization. 3) A number of bacteria and archaea (e.g., Bacillus, Thermoanaerobacter and Thermococcus species) have been isolated as CGTase producers. 6) Many studies on X ray crystallographic structures of CGTases complexed with substrates, products, and inhibitors have revealed that CGTase possesses a substrate binding groove that accommodates at least 7 glucose residues at the donor subsites and 3 at the acceptor subsites (subsite −7 to +3), and contains an aromatic amino acid in a dominant position at the center of the substrate binding groove. 2,7 9) As all known CGTases produce a mixture of CDs, they are subdivided into α , β and γ CGTases based on the major CD product. The majority of CGTases reported are β CGTases while α and γ CGTases are less frequent. γ CD is the most expensive CD due to the low yield of this substance from the current CGTases. Several attempts have been made to enhance the γ CD yield, but no satisfactory results have been obtained to date. 10,11) In addition to the ability of CD synthesis (cyclization reaction), CGTase catalyzes three other reactions: coupling, disproportionation and starch hydrolysis. In the coupling reaction, CD is cleaved and transferred to an acceptor substrate. In the disproportionation reaction and starch hydrolysis reaction, a linear maltooligosaccharide is cleaved and then glycosyl residue transfers to another linear substrate and water, respectively. Thus CGTase is also utilized for the synthesis of glycosides. 12,13) Cyclodextrinase (CDase; EC 3.2.1.54) hydrolyzes CDs much faster than starch and pullulan. 14) Many CDases have been isolated from various bacteria, including Alicy- Abstract: The alkaliphilic soil bacterium Bacillus clarkii 7364 was found to produce cyclodextrin glucanotransferase (CGTase), an enzyme which conver...

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Analysis of the Gene Encoding Cyclomaltodextrinase from AlkalophilicBacillussp. I-5 and Characterization of Enzymatic Properties

Cited by 52 publications

References 28 publications

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

Characterization of Two γ-Cyclodextrin-specific Enzymes from <i>Bacillus clarkii</i> 7364

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Analysis of the Gene Encoding Cyclomaltodextrinase from AlkalophilicBacillussp. I-5 and Characterization of Enzymatic Properties

Cited by 52 publications

References 28 publications

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

Characterization of Two &gamma;-Cyclodextrin-specific Enzymes from <i>Bacillus clarkii</i> 7364

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Characterization of Two γ-Cyclodextrin-specific Enzymes from <i>Bacillus clarkii</i> 7364