• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.225-236
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• 1991

The genes encoding the thermostable $\alpha$-amylase and maltogenic amylase from Bacillus lichenciformis were cloned and expressed in E. coli. The recombinant plasmid pTA322 was found to contain a 3.1kb EcoRI genomic DNA fragment of the thermostable $\alpha$-amylase. The cloned $\alpha$-amylase was compared with the B. licheniformis native $\alpha$-amylase. Both $\alpha$-amylase have the same optimal temperature of $70^{\circ}C$ and are stable in the pH range of 6 and 9. The complete nucleotide sequences of the thermostable $\alpha$-amylase gene were determined. It was composed of one open reading rame of 1,536 bp. Start and stop codons are ATG and TAG. From the amino acid sequence deduced from the nucleotide sequence, the cloned thermostable $\alpha$-amylase is composed of 483 amino acid residues and its molecular weight is 55,200 daltons. The content of guanine and cytosine is $47.46mol\%$ and that of third base codon was $53_41mol\%$. The recombinant plasmid, pIJ322 encoding the maltogenic amylase contains a 3.5kb EcoRI-BamHI genomic DNA fragment. The optimal reaction temperature and pH of the maltogenci amylase were $50^{\circ}C$ and 7, respectively. The maltogenic amylase was capable of hydrolysing pullulan, starch and cyclodextrin to produce maltose from starch and panose from pullulan. The maltogenic amylase also showed the transferring activity. The maltogenic amylase gene is composed of one open reading frame of 1,734bp. Start and stop codons are ATG and ATG. At 2bp upstream from start codon, the nucleotide sequence AAAGGGGGAA seems to be the ribosome-binding site(RBS, Shine-Dalgarno sequence). A putative promoter(-35 and-10 regions) was found to be GTTAACA and TGATAAT. From deduced amino acid sequence from the nucleotide srquence, this enzyme was comosed of 578 amino acid residues and its molecular weight was 77,233 daltons. The content of guanine and cytosine was $48.1mol\%$. The new recombinant plasmid, pTMA322 constructed by inserting the thermostable $\alpha$-amylase gene in the EcoRI site of pIJ322 to produce both the thermostable $\alpha$-amylase and the maltogenic amylase were expressed in the E. coli. The two enzymes expressed from E. coli containing pTMA322 was reacted with the $15\%$ starch slurry at $40^{\circ}C$ for 24hours. The distribution of the branched oligosaccharides produced by the single-step process was of the ratio 50 : 50 between small oligosaccharide up DP3 and large oligosaccharide above DP3.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.273-278
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• 2002

Two-step fed-batch fermentations were carried out to overproduce Bacillus licheniformis maltogenic amylase (BLMA) in recombinant Escherichia coli. The first step was to increase the cell mass by controlling the feeding of a glucose solution, while the second step was designed to improve the amylase expression efficiency by supplementing organic nitrogen sources. The linear gradient feeding method was successfully adopted to maintain the glucose concentration below 0.2 g/l during the fed-batch mode, as effectively minimizing acetic acid formation. When the dissolved oxygen (DO) level became limiting, an accumulation of acetic acid and drastic decrease in specific BLMA productivity were observed. Glucose and organic nitrogen sources consisting of yeast extract and casein hydrolysate were simultaneously supplied in the pH-stat mode to further increase the specific BLMA expression efficiency. An organic nitrogen source consisting of 200 g/1 yeast extract and 100 g/1 casein hydrolysate was found to be the best among the various combinations tested. The feeding of an organic nitrogen source in the second-step fed-batch period was highly beneficial in enhancing the BLMA production. The optimized two-step fed-batch culture resulted in 78 g/l maximum dry cell mass and 443 U/ml maximum BLMA activity, corresponding to 1.5-fold increase in the dry cell mass and 3.7-fold enhancement in BLMA production, compared with the simple fed-batch fermentation.

• Korean Journal of Food Science and Technology
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• v.25 no.6
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• pp.649-654
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• 1993

We investigated the texture of the sweet potato starch gels treated with maltogenic amylase. Effects of branched gluco-oligosaccharides and acorn starch on the texture of the sweet potato starch gel were also investigated. Hardness and cohesiveness of gels were measured by using Instron and sensory evaluation on the gel properties was performed. From the results of the instrumental analysis, it was found that the overall textural properties as Mook could be improved by adding branched glucooligosaccharides, maltogenic amylase or acorn starch to the sweet potato starch gel. As a result, there was a decrease in the cohesiveness of gels while the hardness of gels increased. The sensory evaluation study indicated that the sweet potato starch gels treated with 0.02% maltogenic amylase, or added with 12.5% branched gluco-oligosaccharides, or mixed with 50% acorn starch had preferable quality as Mook.