• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.489-498
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• 2013

A new Bacillus strain degrading starch, named Bacillus sp. UEB-S, was isolated from a southern Tunisian area. Amylase production using solid-state fermentation on millet, an inexpensive and available agro-resource, was investigated. Response surface methodology was applied to establish the relationship between enzyme production and four variables: inoculum size, moisture-to-millet ratio, temperature, and fermentation duration. The maximum enzyme activity recovered was 680 U/g of dry substrate when using $1.38{\times}10^9$ CFU/g as inoculation level, 5.6:1 (ml/g) as moisture ratio (86%), for 4 days of cultivation at $37^{\circ}C$, which was in perfect agreement with the predicted model value. Amylase was purified by Q-Sepharose anion-exchange and Sephacryl S-200 gel filtration chromatography with a 14-fold increase in specific activity. Its molecular mass was estimated at 130 kDa. The enzyme showed maximal activity at pH 5 and $70^{\circ}C$, and efficiently hydrolyzed starch to yield glucose and maltose as end products. The enzyme proved its efficiency for digesting raw cereal below gelatinization temperature and, hence, its potentiality to be used in industrial processes.

• BMB Reports
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• 제37권4호
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• pp.422-428
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• 2004

Raw-starch-digesting $\alpha$-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.251-259
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• 1990

생전분 분해력이 강력한 glucoamylase를 생산하는 균주로서 Asp.usamii IAM 2185를 선정하였다. 밀기울배지에서의 효소생산의 최적 initial pH는 6.0-8.0, 최적 배양온도는 25-$30^{\circ}C$, 최적 배양시간은 72시간이고, 밀기울배지에 ammonuim nitrate와 albumin의 첨가는 효소의 생산을 약간 증가시켰다. 황산암모늄분획, CM-cellulose와 DEAE-cellulose column chromatography에 의하여 효소를 정제하였고, 정제효소의 specific activity는 34.3U/mg.protein, 수율은 10.3 이었다.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.7-13
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• 1992

A moderately thermophilic, alkalophlic and powerful crystalline cellulose-digesting bacterium, Bacillus K-12, was isolated from filter paper wastes and found to be similar to Bacillus circulans or Bacillus pumilis, except for its ability to grow at a moderately high pH and temperature. The isolate grew at a pH ranging from 6 to 10 and at a temperature ranging from 35 to $65^{\circ}C$ and produced a large amount of cellulase components containing avicelase, xylanase, CMCase, and FPase when grown in avicel medium for 5 to 7 days at $50^{\circ}C$. The crude enzyme preparation from the culture broth hydrolyzed xylan, raw starch, pullulan and ${\beta}-1,3$ glucan such as laminarin. Furthermore, the enzyme hydrolyzed crystalline cellulose to cellobiose and glucose and had a broad pH activity curve (pH 6~9). The enzyme was stable up to $70^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.183-188
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• 1992

A soil bacterium which produces raw starch-digesting $\beta$-amylase in culture medium, has been screened from soils. One strain, isolated and identified as Bacillus polymyxa No. 26, was selected as a $\beta$-amylase producing bacterium. Morphological and biological characteristics of the strain were found to be similar to those of a strain belonging to B. polymyxa. The electron microscopic observations of the bacterial vegetative cells and sporulated cells were extensively done to know the corelation between the enzyme synthesis and sporulation. When the bacterium was cultured on the appropriate media (3% dextrin, 0.3% beef extract, 0.5% polypeptone, 1% yeast extract and 0.3% NaCl at pH 7.0 for 4 days) raw starch-digestible $\beta$-amylase was produced extracellularly. This strain produced 130 units of $\beta$-amylase per ml in a culture medium containing 3% dextrin at $30^\circ{C}$. This value is compared to those of other $\beta$-amylase-producing strains. The optimum pH and temperature for crude enzymes were pH 6.5 to 7.0 and $50^\circ{C}$, respectively. The enzymes were stable between pH 5.5 and 9.0 for 30 min at $45^\circ{C}$.

• 한국균학회지
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• 제15권3호
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• pp.175-182
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• 1987

1. $(NH_4)_2SO_4$침 전, DEAE-cellulose column chromatography, CM-Sephadex C-50 및 Sephadex G-75 gel filtration에 의하여 정제된 효소는 조효소보다 18배가 높았으며 회수율은 13.40%였다. 2. 겉보기 분자량은 약 40,000dalton이었다. 3. 최적 pH와 온도는 4.0, $40^{\circ}C$일 때이며, 안정범위는 pH2.0에서 pH5.0, 온도는 $60^{\circ}C$ 이하이었다. 4. 금속이온 첨가에 의한 영향은 $10^{-2}M$ $BaCl_2$첨가시 효소활성이 증가되었으며, $10^{-2}M$ $Pb(NO_3)_2$, $K_3Fe(CN)_6$, $AgSO_4$와 $ZnSO_4$첨가시는 효소활성이 완전히 저해되었다. 5. 각종 유기화합물 첨가에 의한 효소의 영향은 citric acid 첨가시 효소활성이 현저히 저해되었다. 6. 기질 특이성을 보면 dextrin과 glycogen을 기질로 하였을 때는 효소활성이 증가하였으나 saccharose 첨가시에는 현저히 저하되었다. 7. COD 제거율을 측정한 결과는 67에서 68% 정도이었다.

• 한국식품영양과학회지
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• 제32권3호
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• pp.381-387
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• 2003

본 연구에서는 생 전분 분해효소를 이 용한 비 열처리 현미식초 제조방법 확립과 초산발효 중 성분변화를 조사하였다. 초기 알콜농도별, 초기 산도별로 각각 실험한 결과 pH는 초기 알콜농도별 실험치와 초기 산도별 실험치 모두 발효 초기 3.44~4.06에서 발효가 진행됨에 따라 점점 감소하여 총산이 가장 높은 수치를 나타낸 시점에서 각각 2.90~3.44로 가장 낮은 수치를 나타낸 후 증가하는 경향이었다. 총산은 알콜농도가 높을수록 총산함량도 각각 4.23, 5.46, 5.79, 6.50 및 7.18로 점점 높게 나타났으며, 초기 산도별 실험에서는 모두 발효 1일째부터 서서히 증가하여 각각 4.78, 5.88, 5.79및 5.21로 최고치를 나타낸 후 감소하는 경향이었다. 초산 수율은 알콜농도 5%와 6%에서 60%이상으로 높게 나타났으며, 초기 산도 1.0에서 62%로 가장 높게 나타났다. 또한 초산발효과정 중 성분변화를 조사한 결과 pH는 발효초기 3.67에서 발효가 진행됨에 따라 점점 감소하여 3.16으로 나타났으며, 총산은 발효초기 1.02로 발효 1일째부터 총산이 서서히 증가하여 발효 2일에 1.54, 발효 12일에 5.53으로 가장 높은 수치를 나타낸 후 감소하는 경향이었다. 유기산 조성은 oxalic, malic, acetic, citric 및 succinic acid가 검출되었으며, 유기산 중 acetic acid가 가장 높은 함량으로 나타났다. 유리 아미노산은 1,121.09mg%이었으며, ${\gamma}$-aminobutyric acid, $\alpha$-aminoadipic acid, alanine이 주된 아미노산으로 나타났으며, 특히 ${\gamma}$-aminobutyric acid는 539.05 mg%로 아미노산중에서 가장 높은 함량으로 나타났다. 무기질함량은 P와 K함량이 유사하게 높은 함량으로 나타났으며, 다음으로 Mg, Na, Ca순으로 나타났다.

• 한국식품영양과학회지
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• 제32권3호
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• pp.375-380
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• 2003

현미를 이용하여 증자하지 않고 생전분 분해효소를 이용한 알콜발효조건을 모니터링 하였다. 알콜발효에 가장 영향을 미치는 요인변수인 현미분쇄 정도에 따른 입자크기(20, 40, 60 mesh), 효소제 함량(0.1, 0.3, 0.5%), 교반속도(0, 100, 200 rpm)에 대하여 반응표면회귀분석한 결과, 현미분쇄 정도는 35~42 mesh, 효소제함량 0.32~0.43%(w/w) 범위로 나타났으며, 이때 알콜발효의 품질인자인 알콜함량, 총당, 환원당, 아미노산도, 총산 및 pH의 $R^2$는 환원당을 제외한 모든 성분에서 5~l0%이내의 수준에서 유의성이 인정되었다. 또한 최적조건범위 에서 실증실험 결과 pH 4.33, 총산 1.10, 아미노산도 0.51, 환원당 200.50 mg%, 총당 1,039.0 mg%, 알콜함량 10.15%로 RSM기법에 의해 예측된 값과 유사한 값을 나타내어 도출된 회귀식의 신뢰성을 검증할 수 있었다. 또한 최적조건의 알콜발효 중 성분분석 결과 pH는 6.26에서 발효가 진행됨에 따라 점 점 감소하여 발효 종료시 4.34이었으며, 총산은 발효초기 0.15였으며 발효가 진행됨에 따라 다소 증가하였으나 발효종료시 0.20으로 총산의 변화는 크지 않았다. 아미노산도는 발효초기 1.88에서 발효가 진행됨에 따라 감소하는 경향이었으며 발효종료시 0.92었다. 환원당과 총당은 발효가 진행됨에 따라 감소하여 각각 213, 1,077 mg%로 나타났다. 알콜함량은 발효 24시간부터 증가하여 발효 48시간에 10.15%로 최대치를 나타낸 후 감소하였다. 이상의 결과로 생전분분해효소를 이용한 현미 알콜발효조건을 설정할 수 있었으며, 초산발효에 필요한 알콜함량을 얻을 수 있어 생전분을 이용한 현미식초 제조가 가능할 것으로 생각된다.

• 한국식품저장유통학회지
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• 제8권4호
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• pp.412-418
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• 2001

본 연구에서는 현미의 무증자 알콜발효조건을 설정하고자 반응표면분석으로 알콜발효조건을 모니터링하였다. 그 결과 무증자 알콜발효에 적합한 S. cerevisiae GRJ를 선발할 수 있었다. 알콜함량, brix, pH 및 총산에 대한 회귀식의 $R^2$는 각각 0.8828, 0.8409, 0.9431 및 0.9280이였다. 무증자 알콜발효에서 알콜함량의 최대치는 효소제 함량 0.34%(w/w) 및 가수량 350(v/w)의 범위에서 얻을 수 있었다. 최적조건에서 실제 실험값과 예측값들은 유사한 경향을 나타내었다..