• 한국미생물·생명공학회지
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• 제10권4호
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• pp.259-265
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• 1982

Bacillus circulans F-2 $\alpha$-amylase에 의한 각종 oligosaccharide의 분해산물을 조사하고, Bacillus circulans F-2 $\alpha$-amylase, Bacillus amyloliquefaciens $\alpha$-amylase, Rhizopus niveus glucoamylase에 의한 각종 생전분의 분해율을 비교 검토한 결과를 요약하면 다음과 같다. 1. 공시 oligosaccharide중 Bacillus circuians F-2 $\alpha$-amylase는 maltose와 maltotriose를 분해시키지 못하였으나 meltotetraose이상의 oligosaccharide는 분해시켰다. maltotetraose, maltopentaose, maltoheraose, maltoheptaose, maltooctaose중 maltotetraose의 분해가 가장 미약하였다. 2. soluble starch, amylopectin, amylose, oyster glycogen중 oyster glycogen의 분해율이 약간 낮았다. 3. corn starch의 분해율은 진탕반응이 정치반응보다 높았으나 potato starch에 대하여는 효소의 종류에 따라 일정치 않았다. 4. corn, rice, arrowroot, high amylose corn, banana, sago, yam, potato starch에 대하여 Bacillus circulans F-2 $\alpha$-amylase가 Bacillus amyloquefaciems $\alpha$-amylase, Rhizopus niveus glucoamylase보다 월등하게 높은 분해율을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.

• 한국미생물·생명공학회지
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• 제18권4호
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• pp.352-359
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• 1990

분쇄마찰매체효소반응계에서 glucoamylase와 Alpha-amylase의 보완작용에 의한 옥수수 생전분의 효소당화 mechanism을 규명코자, 분리.정제된 상기 효소의 혼합 비율에 따른 당화양상 및 생성당 조성, 전분입자 구조, 생전분입자의 particle size 분포, X-ray 회절양샹의 변화 등을 관찰하였다. 분쇄마찰매체 효소반응계에서도 효소를 분리하여 사용하였을 때보다 glucoamylase와 $\alpha$-amylase를 혼합사용할 경우 당화율이 크게 향상되었으며, 고순도의 glucose 생산에 적합한 두 효소의 혼합비율은 약 1:1-1:2(unit) 로 판명되었다.

• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.1-8
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• 1999

Most carbohydrates exist in nature in an insoluble state, which reduces their susceptibility towards various carbohydrases. Accordingly, they require intensive pretreatment for structural modification to enhance an enzyme reaction. The direct conversion of insoluble carbohydrates has distinct advantages for special types of reaction, especially exo-type carbohydrase; however, its application is limited due to structural constraints. This paper introduces two novel heterogeneous enzyme reaction systems for direct conversion of insoluble carbohydrates; one is an attrition coupled enzyme reaction system containing attrition-milling media for enhancing the enzyme reaction, and the other is a heterogeneous enzyme reaction system using extruded starch as an insoluble substrate. The direct conversion of typically insoluble carbohydrates, including cellulose, starch, and chitin with their corresponding carbohydrases, including cellulase, amylase, chitinase, and cyclodextrin glucanotransferase, was carried out using two proposed enzyme reaction systems. The conceptual features of the systems, their reaction characteristics and mechanism, and the industrial applications of the various carbohydrates are analyzed in this review.

• 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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• 제17권2호
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• pp.136-141
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• 1989

Streptomyces sp. 4M-2의 생전분 분해효소를 황산 암모니움 염석과 이온교환 크로마토그라피 및 Gel 여과 등으로 비활성이 51.22 RSU/mg, 수율 4.5%로 정제할 수 있었다. 정제효소의 분자량은 102,000이었으며 생옥수수 전분에 대한 Km값은 44.44mg/m1 이었다 정제효소의 작용 최적온도는42$^{\circ}C$, pH는 5.5였고 $Ca^{2+}$와 $Ba^{2+}$의 첨가에 의해 효소활성이 증가되었다. 정제효소는 옥수수 amylose를 가장 잘 분해하고 감자전분도 비교적 잘 분해하였다. 이 효소에 의한 옥수수 생전분의 분해산물은 주로 maltose와 maltotriose였고 소량의 glucose와 oligosaccharide도 검출되었으므로 $\alpha$-amylase 계통의 효소로 추정된다.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.109-114
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• 1997

The Oomycete Saprolegnia ferax produces an extracellular $\beta$-amylase, Maximum enzyme yield was attained after 7 days of growth in YNB starch medium (pH 6.5) at 25$\circ$C. The amylase was pu- rified 24-fold by ultrafitration, HPLC DEAE column and HPLC gel filtration. The purfied enzyme was a monomeric glycoprotein with a molecular weight of about 44,000 dalton. The pH and temperature optima were 6.5 and 50$\circ$C, respectively. The enzyme was fairly stable up to 50$\circ$C and at acidic pH region (pH 4.0-7.0). The apparent Km and Vmax values of the enzyme against soluble starch were 0.77 mg/ml and 2,174 $\mu$moles/mg protein, respectively. Amino acid analysis indicated that the enzyme was enriched in alanine, glycine, leucine and acidic amino acid. Starch hydrolysis with the enzyme released maltose but not glucose, whereas maltotriose, Schardinger dextrin ($\alpha$-cyclodextrin) and pullulan were not hydrolysed by the enzyme. The enzyme was inhibited by Schardinger dextrin, p-chloromercuribenzoate(PCMB), CU$^{2+}$' and Hg$^{2+}$. Inhibition of the enzyme by PCMB could be reversed by the addition of cysteine and mercaptoethanol.

• Applied Biological Chemistry
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• 제28권1호
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• pp.1-7
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• 1985

Pseudomonas stutzeri IAM 12097이 생산(生産)하는 Exo-maltotetraohydrolase는 soluble starch, amylose, amylopectin, oyster glycogen, corn, potato, glutinous rice, green banana, arrow root 의 호화전분(糊化澱粉), maltopentaose, maltohexaose, maltoheptaose, maltooctaose를 가수분해(加水分解)하였으나, ${\alpha},{\beta},{\gamma}-cyclodextin$, sucrose, raffinose, pullulan, maltose, maltotriose, maltotetraose는 분해(分解)하지 못하였다. 소당류중(少糖類中) maltohexaose를 가장 잘 분해(分解)하였으며, 각종기질(各種基質)의 주분해산물(主分解産物)은 maltotetraose였다. pullulanase의 혼용(混用)으로 호화전분(糊化澱粉)의 분해율(分解率)은 증가(增加)되었으나 생전분(生澱粉)에 대(對)하여는 혼용효과(混用效果)를 인정(認定)할 수 없었고 생전분중(生澱粉中)에서 corn starch가 가장 잘 분해(分解)되며 raw potato starch, raw arrow root starch, raw high amylose corn starch의 분해(分解)는 미약(微弱)하였다.

• Applied Biological Chemistry
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• 제40권5호
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• pp.395-399
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• 1997

아밀로오스 함량이 다른 옥수수전분을 2.2N HCl로 산처리하여 산가수분해 특성 및 효소저항전분의 수율을 비교하였다. 산 가수분해경향은 아밀로오스 함량이 0%인 Amioca는 7일을 경계로, 그외의 전분은 4일을 경계로 2단계로 진행되었으며, 산가수분해 속도상수는 초기에 $4.01{\sim}9.21{\times}10^{-3}hr^{-1}$, 후기에는 $1.60{\sim}5.01{\times}10^{-3}hr^{-1}$로 아밀로오스 함량이 낮을수록 옥수수전분의 가수분해 속도가 빨랐다. 생전분의 X-선 회절양상은 아밀로오스 함량이 적은 Amioca, PFP, CMS(commercial maize starch)는 A형 , 고아밀로오스 함량인 Amaizo 5, Amylomaize VII은 B형의 전형적인 결정형을 보였으며, 산처리 후에도 모든 시료의 결정형은 그대로 유지하였으나 상대적인 결정화도는 증가하였다. 효소저항전분의 수율은 Amioca, CMS, Amylomaize VII이 각각 1.8%, 20.8%, 37.9%로 아밀로오스 함량이 높을수록 수율도 증가하였으며, 가열-냉각 횟수를 4회 반복한 후 분리한 산처리 한 옥수수전분의 효소저항전분의 수율은 1일 산처리한 CMS, Amylomaize VII이 각각 4.5%, 29.1%였으며 7일 산처리시에는 1.5%와 19.4%로 산처리에 의해 효소저항전분의 수율이 감소하였다.

• BMB Reports
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• 제35권6호
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• pp.568-575
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• 2002

An $\alpha$-amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipiation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and $60^{\circ}C$. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had $\alpha$-anomeric forms, as determined by $^1H$-NMR. This maltose-forming $\alpha$-amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62%, repectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.