• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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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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• 제12권2호
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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.

• 한국식품과학회지
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• 제25권6호
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• pp.649-654
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• 1993

효소를 이용하여 전분을 수식하여 묵의 텍스쳐 특성을 개선하고 아울러 기능성 탄수화물을 첨가시켜 불성을 개선하고 건강식품화하고자 하였다. 전분의 분자수식을 위하여 maltogenic amylase를 이용하였으며 고구마 전분의 구조를 modification시켜 묵을 제조하고 또한 분지 글루코올리고당과 도토리 전분을 혼합하여 10% 전분겔을 제조한 후 Instron을 사용한 기계적 검사와 관능검사를 병행하여 텍스쳐 특성을 시험하였다. 고구마 전분에 maltogenic amylase를 0.02% 첨가하였을 때 텍스쳐 개선효과가 가장 켰으며 묵의 품질이 우수하였다. 이는 효소에 의해 긴 아밀로오스와 아밀로펙틴이 묵이 형성될 수 있는 길이로 잘라졌기 때문으로 생각된다. Bacillus licheniformis maltogenic amylase(BLMA)를 이용하여 제조한 분지글루코올리고당을 첨가한 경우, 12.5%를 첨가하였을 때 텍스쳐 특성이 가장 향상되었다. 도토리 전분을 혼합하여 전분겔을 제조한 경우에는 50%를 혼합하였을 때 가장 텍스쳐 특성이 향상되었고 100% 도토리만으로 제조한 전분겔보다 수응력이 우수하였다.