• 한국식품조리과학회지
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• 제12권4호
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• pp.522-534
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• 1996

The present study was conducted to investigate quality characteristics of Nochi made with malted barley flour with (Cl) and without hull (C2), comparing with Nochi that was treated with different sources of commercial amylases. There was higher level of moisture content (18.4%) in Nochi treated with fungal ${\alpha}$-amylase (FU) comparing with the other Nochi samples. However, Nochi that was treated with bacterial ${\alpha}$-amylase and ${\beta}$-amylase (BA-${\beta}$) had the lowest level of moisture content (11.2%). Nochi samples which were treated with thermostable ${\alpha}$-amylase and fungal ${\alpha}$-amylase(TE-FU) were different from traditional Nochi samples in mechanical characteristics. According to the results of sensory evaluation, Cl was similar to C2 except in cohesiveness and malt flavor. TE-FU and Bh-${\beta}$ were not different from traditional Nochi in cohesiveness, sweetness and overall desirability.

• 미생물과산업
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• 제19권2호
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• pp.34-41
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• 1993

Industrial strain improvement is concerned with developing or modifying microorganisms used in production of commercially important fermentation products. The aim is to reduce the production cost by improving productivity of a strain and manipulating specific characteristics such as the ability to utilize cheaper raw materials or resist bacteriophages. The traditional empirical approach to strain improvement is mutation combined with selection and breeding techniques. It is still used by us to improve the productivity of organisms in amino acids, organic acids and enzymes production. The breeding of high L-lysine-producing strain Au112 is one of the outstanding examples of this approach. It is a homoserine auxotroph with AEC, TA double metabolic analogue resistant markers. The yield reaches 100 g/l. Besides, the citric acid-producing organism Aspergillus niger, Co827, its productivity reaches the advanced level in the world, is also the result of a series mutations especially with $^60Co{\gamma}$-radiation. The thermostable .alpha.-amylase producing strain A 4041 is the third example. By combining physical and chemical mutations, the strain A 4041 becomes an asporogenous, catabolite derepressed mutant with rifamycin resistant and methionine, arginine auxotroph markers. The .alpha.-amylase activity reaches 200 units/ml. The fourth successful example of mutation in strain improvement is the glucoamylase-producing strain Aspergillus niger SP56, its enzyme activity is 20,000 units/ml, 4 times of that of the parental strain UV-11. Recently, recombinant DNA approach provides a worthwhile alternative strategy to industrial strain improvement. This technique had been used by us to increase the thermostable .alpha.-amylase production and on some genetic researches.

• Applied Biological Chemistry
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• 제41권1호
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• pp.18-22
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• 1998

Bacillus licheniformis를 화학적 돌연변이를 시켜 내열성 ${\alpha}-amylase$ 고생산성 변이주 SK-5를 얻었다. 변이주는 모균에 비하여 약 50배 정도의 ${\alpha}-amylase$를 생산하였으며, 그 모양이 가늘고 길이가 길어졌고, 성장속도가 감소되었다. 이 효소의 유전적 변화를 분석하기 위하여 변이주 SK-5로부터 ${\alpha}-amylase$ 유전자 염기배열을 결정한 결과 구조유전자의 염기배열은 동일하였으나 promoter 지역에서 일부 변이가 일어난 것이 확인되어 이것이 부분적으로 효소생산성 증가에 영향을 미칠 것으로 여겨진다. SK-5의 ${\alpha}-amylase$ 생산성이 높기 때문에 이의 배양상층액으로부터 열처리와 황산암모늄 침전 후 한 단계의 hydroxyapatite 컬럼을 사용하여 순수하게 정제된 ${\alpha}-amylase$를 얻을 수 있었다. 변이에 따른 세포외 단백질분해효소의 영향을 검증하기 위하여 SK-5 배양액을 시간별로 준비하여 Western blot으로 분석한 결과 변이주에서 분비되는 ${\alpha}-amylase$의 구조에 변화가 없음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.2000-2003
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• 2006

A gene (GenBank AF096282) coding for a $\alpha$-glucosidase (TcaAG, EC 3.2.1.20) from Thermus caldophilus GK24 was expressed in Saccharomyces cerevisiae, a generally recognized as safe (GRAS) host. The thermostable $\alpha$-glucosidase was produced inside of the GRAS host at 0.04 unit/mg-dry cell by the constitutively expressing ADH1 promoter and at 1.2 unit/mg-dry cell by the inductively expressing GALl0 promoter, respectively. No $\alpha$-glucosidase activities were found in the medium when the MF-alpha signal sequence from S. cerevisiae or $\alpha$-amylase signal sequence from Aspergillus oryzae were fused before the $\alpha$-glucosidase gene for the secretion.

• BMB Reports
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• 제34권4호
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• pp.347-354
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• 2001

$\alpha$-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and $90^{\circ}C$, and was stable from pH 6.0 to 85 and up to $90^{\circ}C$. The enzyme had a half-life of 85 minutes at $90^{\circ}C$. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of $\alpha$-1,6-glucosidic linkages of isomaltosaccharides and panose, $\alpha$-1,3-glycosidic bond of nigerose and turanose, and $\alpha$-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the $\alpha$-amylase family. We suggest that this monomeric, thermostable, and broad-acting $\alpha$-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel $\alpha$-glucosidase.

• 한국미생물·생명공학회지
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• 제4권3호
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• pp.91-97
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• 1976

고온성 방선균으로부터 내열성 $\alpha$-Amylase의 생산과 그 이용 가능성을 검토하기 위하여 토양시료로부터 $\alpha$-Amylase 생산능력이 극히 우수한 구주를 분리하여 분리균의 몇가지 균학적 성질내지는 효소생산을 위한 배양조건을 조사하여 전보에 발표하였으며 본보에서는 공시방선균이 생산하는 $\alpha$-Amylase의 효소학적 일반성질을 검토하여 다음과 같은 결과를 얻었다. 1) 본효소의 최적활성 pH는 6.5이었으며 최적변성온도는 55$^{\circ}C$~$65^{\circ}C$이었다. 2) 본효소의 안정 pH범위는 7.0~8.0에 위치 하였으며 3) 활성에 대한 금속 ion의 영향은 $Ca^{＋＋}$ 및 $Na^{＋＋}$ ion에 의하여 촉진되었으나 중금속 ion인 Fe$^{＋＋}$ Cu$^{＋＋}$ ion등은 현저히 조해함을 보였으며 열안정성에 대한 금속 ion의 효과는 $Ca^{＋＋}$ ion에 의해 증가했다. 4) 본 순소의 Km value는 2.17$\times$$10^{-4}$g per $m\ell$이었으며 5) Activation energy는 12,000$\pm$580 ㎈ per mole 이었다.

• 한국식품과학회지
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• 제32권3호
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• pp.618-628
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• 2000

열처리 덱스트린을 내열성 ${\alpha}-amylase$로 가수분해함으로써 난소화성 텍스트린 I을 제조하였다. 황색 덱스트린으로부터 제조한 난소화성 덱스트린 I의 난소화성 획분과 식이섬유 함량의 평균치는 각각 50%와 25%였다. 또한, 열처리 덱스트린의 효소반응액으로부터 에탄올 처리에 의해 glucose를 포함하는 저분자의 당류를 제거함으로써 난소화성 덱스트린 II를 제조하였다. 초기 효소반응액의 80% 이상의 glucose와 maltose가 제거되어 난소화성 획분과 식이섬유 함량이 증가하였다. ${\alpha}-amylase$ 및 amyloglucosidase에 의한 황색 덱스트린의 가수분해액의 에탄올 침전으로 제조된 난소화성 덱스트린이 다른 효소 조합보다 높은 함량의 난소화성 획분 및 식이섬유를 나타내었으며, 각각 평균 83.6%와 62.8%였다. 따라서, 전분 가수분해 효소에 저항성을 갖는 난소화성 덱스트린은 열처리 덱스트린으로부터 용이하게 제조될 수 있으며, 수용성 식이섬유로서의 생리효과를 발휘할 수 있을 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1242-1248
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• 2007

Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to ${\alpha}$-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the ${\alpha}$-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at $80^{\circ}C$ and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life ($t_{1/2}$) values of 10 min at $90^{\circ}C$, despite the high similarity to ${\alpha}$-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermo stability. The presence of $Ca^{2+}$ seemed to be critical, significantly changing $t_{1/2}$ at $90^{\circ}C$ to 153 min by the addition of 0.5 mM $Ca^{2+}$. On the other hand, the thermostability was not enhanced by the addition of $Zn^{2+}$ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermo stability, indicating that the residues involved in metal binding is very critical for the thermostability.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• 한국식품과학회지
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• 제41권4호
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• pp.369-373
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• 2009

전분액화에 호화찰옥수수전분가 쌍축형 압출성형의 원료로 직접 사용되었다. 그 액화 효과와 체류시간분포를 분석하였다. 내열성 ${\alpha}$-amylase(Bacillus licheniformis로부터 분리)를 함께 첨가하여 사용하였다. 전분액화는 환원당측정, gel permeation chromatography(GPC), 주사전자현미경을 통하여 분석하였다. 배럴온도와 수분함량이 높을수록 환원당 함량은 증가하였고, 호화전분의 경우 생전분보다 더 많은 환원당이 생성되었다. GPC에서 호화전분의 사용에 의한 저분자화 효과는 뚜렷하지 않았으나, 효소 첨가없이 압출성형하는 경우에는 일부 효과가 있는 것으로 나타났다. 주사전자현미경의 미세구조에서는 호화전분을 쓸 경우 그 표면이 더 불규칙하고 침식되는 것을 관찰할 수 있었다. 종합적으로 호화전분의 사용이 전분액화에 효과가 있음을 알 수 있었다. 체류시간분포에서는 호화전분을 원료로 할 경우 그 분산도가 커지는 것으로 나타났다. 이는 호화전분은 압출성형의 흐름이 원만하지 않은 것을 의미하였으나, 배럴온도와 수분함량을 증가시킴에 따라 그 분산도를 감소시킬 수 있음을 알 수 있었다.