Purification and characterization of a thermostable α-glucosidase from aBacillus subtilis high-temperature growth transformant

Krohn, Bradley M.; Lindsay, James

doi:10.1007/bf02091954

Cited by 6 publications

(2 citation statements)

References 14 publications

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“…The CDases from other thermophilic sources, B. subtilis H-17 and T. ethanolicus 39E, have optimum temperatures of 65 to 68°C (24) and 65°C (38,40), respectively. The strain B1001 CDase exhibited a half-life of 2 h at 90°C and the hydrolytic activity was maintained in the presence of substrates even after 18 h at 90°C (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…After the first report of the CDase from Bacillus maceranse (8), CDases from various microorganisms such as Bacillus coagulans (23), Bacillus sphaericus E-244 (34,35), Clostridium thermohydrosulfuricum strain 39E (recently reclassified as Thermoanaerobacter ethanolicus strain 39E) (38,40), alkalophilic Bacillus sp. (50), Bacillus subtilis strain H-17 (24,25), Flavobacterium sp. (3), B. sphaericus strain ATCC 7055 (15), Klebsiella oxytoca strain M5a1 (11,12), and alkalophilic Bacillus sp.…”

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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: Discussionmentioning

confidence: 99%

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confidence: 99%

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

et al. 2001

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Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

Krohn

Lindsay

1993

Current Microbiology

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The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.

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Development of a strategy for the screening of α-glucosidase-producing microorganisms

et al. 2020

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Purification and characterization of a thermostable α-glucosidase from aBacillus subtilis high-temperature growth transformant

Cited by 6 publications

References 14 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

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

Development of a strategy for the screening of α-glucosidase-producing microorganisms

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