• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.161-168
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• 1986

A 1.95Kb Sau3Al fragment coding for $\alpha$-amylase from Bacillus amyloliquefaciens was isolated by the shotgun method using Escherichia coli as a host. The genome of Bacillus amyloliquefaciens was partially digested with the restriction endonuclease Sau3Al and joined to plasmid YRp7 cleaved with the restriction endonuclease BamHI. The $\alpha$-amylase gene present in a 1.95Kb insert was stably maintained and expressed in Escherichia coli. The amount of $\alpha$-amylase activity produced by Escherichia coli containing the hybrid plasmid pEA24 was about 65% of the activity produced by the donor Bacillus amyloliquefaciens strain. The properties of $\alpha$-amylase produced by Escherichia coli were very similar to those produced by Bacillus amyloliquefaciens as based on optimum temperature, pH, and effect of CaCl$_2$ concentration. About 70% of the $\alpha$-amylase produced by Escherichia coli was localized in the periplasmic space, whereas the remaining enzyme was localized in the inner part of the cell.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.479-485
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• 1986

A 5200 basepair DNA fragment containing the Bacillus amyloliquefaciens amyE gene, encoding liquefying $\alpha$-amylase (1,4-$\alpha$-1)-glucan glucanohydrolase, EC 3.2.1.1), has been inserted into BamHI site of the pUB110 and the hybrid plasmid was designated as pSKS3. The pSKS3 was transformed into the Bacillus subtilis KM2l3 as a host which is a saccharifying $\alpha$-amylase deficient mutant of Bacillus subtilis NA64, and the plasmid in the transformed cell was expressed $\alpha$-amylase production and kanamycin resistance. The $\alpha$-amylase production of the transformed cell was reduced to one fifth of that of the donor strain. The Bacillus subtilis KM2l3 tarring pSKS3 indicated that the amyE gene product is a polypeptide which has the same electrophoretic mobility with that of the Bacillus amyloliquefaciens, but different from the saccharifying $\alpha$-amylase of Bacillus subtilis NA64. It means that the amyE gene of pSKS3 originales from the Bacillus amyloliquefaciens.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.563-568
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• 2010

The Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the $\alpha$-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant $\alpha$-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type $\alpha$-amylase, the mutant $\alpha$-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant $\alpha$-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the $\alpha$-amylase activity. Interestingly, the mutant $\alpha$-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.358-362
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• 1988

Aqueous two-phase fermentation system was tested for the overproduction of extracellular enzyme through $\alpha$-amylase fermentation by Bacillus amyloliquefaciens. By employing aqueous two-phase system $\alpha$-amylase activity showed 25% increase compared to the result using regular medium and no deactivation of the enzyme was observed. The presence of polyethylene glycol was observed to promote the enzyme production, while to inhibit the growth of the microorganism. It is recommended that polyethylene glycol be added during the log-growth phase and dextran be added after the enzyme activity reaches Its maximum for efficient $\alpha$-amylase fermentation and in situ recovery of the enzyme.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.209-212
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• 1986

$\alpha$-Amylase gene of Bacillus amyloliquetaciens was cloned on plasmid YEp13, S. cerevisiae-E. coli shuttle vector. Hybrid plasmid pTG17, carrying $\alpha$-amylase gene of B. amyloliquefaciens, was transformed to E. coli and the expression of it in yeast was investigated. This plasmid was unstable in E. coli and produced two minor plasmids, pTG17-1 and PTG17-2, which resulted from the segregation of it. Transformant of S. cerevisiae MC16 with pTG17-1 plasmid was not appeared on SD medium because of the Leu2 gene defection. S. cerevisiae could be transformed by the hybrid plasmid, and $\alpha$-amylase activity of the yeast transformant was detected by somogyi-Nelson method and agar diffusion method.

• Applied Biological Chemistry
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• v.24 no.4
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• pp.252-259
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• 1981

The effects of tunicamycin (TM) on the growth and ${\alpha}-amylase$ productivity of B. amyloliquefaciens K were studied. The minimal growth inhibitory concentration was $0.25{\mu}\textrm{g}/m\ell$ and its ${\alpha}-amylase$ was stable up to $50^{\circ}C$. When the saking culture with $1{\mu}\textrm{g}/m\ell$ of Tunicamycin caused the change of cell shape from form rod to irregular circular form and the mycelium lysis. the grow th of His-, $TM^{\tau}$ mutant obtained by treatment of TM and ultraviolet ray was similar to that of the parent strain, but the productivity of ${\alpha}-amylase$, protease, and RNase was lower than that of the parent.

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.133-139
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• 2012

A bacterium, identified as $Bacillus$ $amyloliquefacies$, CNL-90 using 16S rDNA analysis, was isolated from malt grain. The optimal activities of its ${\alpha}$-amylase and protease were observed at pH 6 and $60^{\circ}C$, and at pH 6 and $50^{\circ}C$, respectively although their activities remained stable at pH 7 and $40^{\circ}C$for ${\alpha}$-amylase and at pH 7 and $50^{\circ}C$ for protease. After solid-state fermentation of $B.$ $amyloliquefacies$, CNL-90 on wheat bran for 72hr or 144hr, the ${\alpha}$-amylase and protease activities were 170,000 and 290,000 units/kg, and 290,000 and 310,000 units/kg, respectively. The viable bacterial cell counts were $1.5{\times}10^9$ CFU/g and $2.2{\times}10^9$ CFU/g at 72hr and 144hr of the solid-state fermentation, respectively. A feeding trial with a total of 127 piglets was also conducted. The animals were divided into two groups: an experimental group fed with the fermented product (63 piglets) and a control group (64 piglets). The growth rate of the experimental group was 6.66% higher than that of the control group (P<0.05). The results of this study indicate that the ${\alpha}$-amylase and protease from $B.$ $amyloliquefacies$, CNL-90 can be used for industrial applications due to their activity in production of carbohydrate hydrolysates.

• Journal of Microbiology and Biotechnology
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• v.22 no.5
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• pp.592-599
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• 2012

Bacillus licheniformis ${\alpha}$-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.259-265
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• 1982

These experiments were conducted to investigate the hydrolysis products on the various oligosaccharides of Bacillus cirulans F-2 $\alpha$-amylase, and the hydrolysis rate on the various raw starches of Bacillus circulans F-2 $\alpha$-amylase, Bacillus amylotiquefaciens $\alpha$-amylase and Rhizopus niveus glucoamylase. The results obtained were as follows : 1. Maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose were hydrolyzed, but maltose and maltotriose were not hydrolyzed by Bacillus circulans F-2 $\alpha$-amylase. Among maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose, especially maltotetraose was hydrolyzed weakly by Bacillus circulans F-2 $\alpha$-amylase. 2. The Hydrolysis rate of oyster glycogen was slightly lower than soluble starch, amylose and amylopectin. 3. The hydrolysis rate of com starch was higher in shaking incubation than in stationary incubation, but the hydrolysis rate of potato starch was not definite according to kinds of enzyme. 4. On com, rice, arrowroot, high amylose corn, banana, sago, yam and potato starch, Bacillus circulans F-2 $\alpha$-amylase exhibited a remarkably higher hydrolysis rate than Bacillus amyloquefaciems $\alpha$-amylase and Rhizopus niveus glucoamylase.