• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.9
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• pp.1388-1393
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• 2014

To enzymatically prepare amylopectin cluster (APC), cyclodextrin glucanotransferase (CGTase I-5) and its mutant enzyme from alkalophilic Bacillus sp. I-5 were employed, after which the hydrolysis patterns of CGTase wild-type and its mutant enzyme toward amylopectin were investigated using multi-angle laser light scattering. CGTase wild-type dramatically reduced the molecular weight of waxy rice starch at the initial reaction, whereas the mutant enzyme degraded waxy rice starch relatively slowly. Based on the results, the molecular weight of one cluster of amylopectin could be about $10^4{\sim}10^5g/mol$. To determine production of cyclic glucans from amylopectin, matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed. CGTase I-5 produced various types of cyclic maltooligosaccharides from amylopectin, whereas the mutant enzyme hardly produced any.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.811-819
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• 1999

The ${\beta}-CGTase$ gene of alkalophilic Bacillus firmus var. alkalophilus was cloned into E. coli using $pZErO^{TM}-2$ as a vector. The cloned gene encoded a total of 710 amino acid residues consisting of 674 amino acids of the matured protein and 36 amino acids of the signal peptide, including 20 amino acids from the lacZ gene in the vector. Although the cloned ${\beta}-CGTase$ gene did not contain the promoter and start codons, it was expressed by the lac promoter and lacZ start codon in the $pZErO^{TM}$ vector. A comparison was made with the amino acid sequence and ten other CGTases from Bacillus sp. Also, ten highly conserved regions, which are important amino acid residues in catalysis of CGTase, were identified. The lac promoter used for expression of the ${\beta}-CGTase$ gene was induced constitutively in recombinant E. coli even without IPTG possibly because of a lack of the lacI gene in both host and vector, repressing the lacZ gene in the lac operon. Its expression was catabolically repressed by glucose, however, its repression was reduced by soluble starch, mainly because of the extremely high increase of the cAMP level. ${\beta}-CGTase$ can be overproduced in the recombinant E. coli by maintaining intracellular cAMP levels mostly through the intermittent feeding of glucose during cultivation.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.