• 한국미생물·생명공학회지
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• 제9권3호
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• pp.159-164
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• 1981

부분적 항상성변이주인 Bacillus megaterium (KFCC 10029)가 생산하는 페니실린 아미다제를 예로하여 강화된 $Ca^{++}$-alginate gel에 의한 포괄방법을 이용하는 효소 고정화 방법을 제시하였다. 발효액으로 부터 celite 흡착법에 의해 효소를 분리한 후 alginate의 gellatin용액에 혼합하고 $Ca^{++}$ 용액에서 응고시키고 glutaraldehyde로 처리하여 성형하였다. 이렇게 하여 얻은 고정화효소의 최적 pH 및 온도는 각각 8.0과 6$0^{\circ}C$였다. Km value 와 6-APA 및 페닐초산에 의한 저해 상수는 각각 2.6mM, 7.4mM, 21.2mM이었다. Gel의 증가된 물리적 강도 때문에 반응조 조작중 흡착효소의 유실을 성공적으로 없앨수 있었다. 관형식 반응조에서의 고정화 효소의 반감기는 4$0^{\circ}C$와 3$0^{\circ}C$에서 각각 6일 및 30일이었으며, 이것은 흡착효소와 비교해 볼 때 6-8배의 증가치이다. 결론적으로 alginate gel 포괄방법에 의한 효소고정화 방법에 있어, 본 연구에서 개발된 개량된 방법을 사용함으로써 고정화효소의 물리적 강도 및 안정도를 크게 증가시킬 수 있었다.

• 한국식품과학회지
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• 제27권1호
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• pp.86-91
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• 1995

알긴산이 가지는 여러가지 물성적 특성 및 기능적 특성으로 식품에 널리 이용되고 있으나 상핀 점도와 용해도 때문에 그 이용 범위가 제한적이다. 이러한 알긴산을 보다 광범위하게 식품에 이용하기 위해서 알긴산의 특성을 어느 정도 유지하면서 용해도를 높이고, 점도를 저하시킨 부분 분해 알긴산의 제조와 특성을 실험하였다. 사용된 알긴산 분해효소는 pH 8.0, 온도 $37^{\circ}C$, 0.5 M NaCl 첨가 조건에서 최대 분해 활성을 나타내었다. 기질과 반응하는 부분 정제 효소(220.4 g/ml)의 농도는 2%(v/v)가 적절하였다. 반응 알긴산 농도에 관계없이 반응시간 10분으로 알긴산이 가지고 있는 점도 80%정도가 저하되었고, 60분 이내에 90% 이상의 점도가 떨어졌다. 분해에 의한 환원당 생성량을 측정하여 본 결과, 반응 60분까지는 큰 변화가 없었으나, 그 이후에는 환원당이 크게 증가하였다. 분해 시간에 따라 얻어진 알긴산들의 가용 농도는 반응 알긴산의 농도가 1.0%와 1.5%의 경우 10분 정도의 분해만으로도 10% 농도까지 용해할 수 있었고, 반응 알긴산 농도가 2.0%인 경우는 10분간의 분해로 7%까지 녹일 수 있었다. 알코올 침전도를 알아본 결과, 2.5% 시판 알긴산은 알코올 농도 3%일 때 침전을 시작하여 알코올 농도 30%일 때 22%, 알코올 농도 50%에서 대부분의 알긴산이 침전하였다. 부분 분해 알긴산의 경우는 알코올 농도 30%일 때 침전하기 시작하여 알코올 농도 70%일 때 85% 정도의 알긴산이 침전되었다. $Ca^{2+}$이온과의 반응성은 시판 알긴산과 마찬가지로 종류에 관계없이 부분 분해 알긴산의 농도가 높아짐에 따라 반응성도 커지는 것으로 나타났다.

• 한국미생물·생명공학회지
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• 제48권1호
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• pp.12-23
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• 2020

Edible films containing antimicrobial agents can be used as safe alternatives to preserve food products. Essential oils are well-recognized antimicrobials. However, their low water solubility, volatility and high sensitivity to oxygen and light limit their application in food preservation. These limitations could be overcome by embedding these essential oils in complexed product matrices exploiting the encapsulation efficiency of β-cyclodextrin. This study focused on the maximization of β-cyclodextrin production using cyclodextrin glucanotransferase (CGTase) and the evaluation of its encapsulation efficacy to fabricate edible antimicrobial films. Response surface methodology (RSM) was used to optimize CGTase production by Brevibacillus brevis AMI-2 isolated from mangrove sediments. This enzyme was partially purified using a starch adsorption method and entrapped in calcium alginate. Cyclodextrin produced by the immobilized enzyme was then confirmed using high performance thin layer chromatography, and its encapsulation efficiency was investigated. The clove oil/β-cyclodextrin inclusion complexes were prepared using the coprecipitation method, and incorporated into chitosan films, and subjected to antimicrobial testing. Results revealed that β-cyclodextrin was produced as a major product of the enzymatic reaction. In addition, the incorporation of clove oil/β-cyclodextrin inclusion complexes significantly increased the antimicrobial activity of chitosan films against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Escherichia coli, and Candida albicans. In conclusion, B. brevis AMI-2 is a promising source for CGTase to synthesize β-cyclodextrin with considerable encapsulation efficiency. Further, the obtained results suggest that chitosan films containing clove oils encapsulated in β-cyclodextrin could serve as edible antimicrobial food-packaging materials to combat microbial contamination.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.224-229
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• 2004

토양으로부터 강력한 CAH activity를 갖는 균주를 분리하여, 생화학적, 배양학적, 전자현미경 동정을 한 결과, Bacillus sphaericus로 확인되었으며, 이를 B. sphaericus 366M-9로 명명하였다. 또한 이 균으로부터 최초로 cephalosporin-C deacetylase(CAH)를 분리 정제하였다. 정제수율은 약 7.5% 였으며, B. sphaericus 366M-9에서 분리한 CAH-9의 최적활성 온도는 $50^{\circ}C$였으며, 효소안정 온도구간은 30～$50^{\circ}C$이다. 또한 최적 활성 pH는 7.0이었으며, 효소안정 pH구간은 pH 6.0～8.0으로 90% 이상의 잔존 활성도를 나타내었다. 효소반응속도에 미치는 기질의 영향에서는 $K_{m}$ 값은 0.87 mM 이며,$ V_{max}$는1.22 unit/ml이었다.다.

• Bulletin of the Korean Chemical Society
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• 제34권8호
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• pp.2408-2412
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• 2013

We demonstrate herein the synthesis and modification of magnetic nanoparticles and its use in the immobilization of the lipase. Magnetic $Fe_3O_4$ nanoparticles (MNPs) were prepared by simple co-precipitation method in aqueous medium and then subsequently modified with tetraethyl orthosilicate (TEOS) and 3-aminopropyl triethylenesilane (APTES). Silanization magnetic nanoparticles (SMNP) and amino magnetic nanomicrosphere (AMNP) were synthesized successfully. The morphology, structure, magnetic property and chemical composition of the synthetic MNP and its derivatives were characterized using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) analysis, X-ray diffraction, superconducting quantum interference device (SQUID) and thermogravimetric analyses (TGA). All of these three nanoparticles exhibited good crystallization performance, apparent superparamagnetism, and the saturation magnetization of MNP, SMNP, AMNP were 47.9 emu/g, 33.0 emu/g and 19.5 emu/g, respectively. The amino content was 5.66%. The AMNP was used to immobilize lipase, and the maximum adsorption capacity of the protein was 26.3 mg/g. The maximum maintained activity (88 percent) was achieved while the amount of immobilized lipase was 23.7 mg $g^{-1}$. Immobilization of enzyme on the magnetic nanoparticles can facilitate the isolation of reaction products from reaction mixture and thus lowers the cost of enzyme application.

• Journal of Applied Biological Chemistry
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• 제42권1호
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• pp.7-11
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• 1999

An extracellular chitinase of the selected strong antifungal microorganism, Serratia sp. 3095, was purified by salting out, affinity adsorption, Sepadex G-100 gel fitration, Sepadex G-75 gel fitration and DEAE Sepadex A-50 chromatography. The molecular weight of the purified chitinase was estimated to be 62,000 dalton by SDS-PAGE. Optimal pH and temperature of the chitinase were pH 7.5 and 45, respectively. The enzyme retained more than 80% of the activity between pH 5.5 and pH 10.5, and below $50^{\circ}C$ but was unstable above $60^{\circ}C$, below pH 5.0. The activity of the chitinase was inhibited about 60% by $Sn^{2+}$, 40% by $Hg^{2+}$ and $Ag^+$, 70% by AHA, 40% by iodoacetate, 35% by thiourea and p-CMB, but stabilized by SDS. $K_m$ value of the purified chitinase was 3.68 mg/ml for colloidal chitin. The chitinase from Serratia sp. 3095 showed antifungal activity to Fusariurm solani.

• 대한전기학회:학술대회논문집
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• 대한전기학회 1999년도 추계학술대회 논문집 학회본부 C
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• pp.984-986
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• 1999

In the case of immobilizing of glucose oxidase in organic polymer using electrosynthesis, the glucose oxidase obstructs charge transfer and mass transport during the film growth. This may lead to short chained polymer and make charge-coupling weak between the glucose oxidase and the backbone of the polymer. That is mainly due to insulating property and net chain of the glucose oxidase. Such being the case, it is useless to increase in amount of glucose oxidase more than reasonable in the synthetic solution. We establish by means of qualitative analysis that amount of immobilized glucose oxidase can be improved by adding a hole ethyl alcohol in the synthetic solution. As ethyl alcohol was added by 0.1mol $dm^{-3}$ in the synthetic solution, the faradic impedance of resultant electrode was increased about five times as much as the case of ethyl alcohol free in the solution, and mass transport was limited more than over. That is due to insulating property and net chain of the glucose oxidase. Moreover, in ultraviolet spectra of the synthetic solution, the adsorption peak at 285nm corresponding to glucose oxidase was decreased. It suggests increase in amount of immobilized glucose oxidase.

• 한국염색가공학회지
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• 제6권3호
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• pp.15-26
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• 1994

Cotton fabrics were treated with the cellulase which is an enzyme to decompose cellulose and its actional mechanism is known. The optimum condition of the cellulase to the cotton fabrics and the weight losses, tensile strengths of the treated cotton fabrics were also obtained. The cellulase performs a specific catalytic action on the ${\beta}-1$, 4-glucosidic bonds of the cellulose molecules and hydrolyzes them. For that reason, the negative surface charges of the cotton fabrics were increased by additional generation. of hyrdoxyl groups. The increased surface charges cause the decrease of dye adsorption by inhibiting the approach of the anions of direct dyes. But, it was overcome by the use of enough amount of salt, it means that sodium ions of the salt neutralize the almost all of negative charges of the cotton fabrics. The improvement of the water absorbency is also due to the increased hydroxyl groups In addition, their handles including the mechanical properties were measured and caculated by KES system which is a measuring apparatus that numerizes and objectificates human's feeling, especially touch. As the results, we knew that KOSH(stiffness) and FUKURAMI(fulness & softness) were decreased and that NUMERI(smoothness) was increased.

• 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.

• 멤브레인
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• 제16권1호
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• pp.39-50
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• 2006

실리카 입자를 기공 형성제로 사용하여 다공성 키토산 및 키틴 막을 제조하였다. 다공성 막의 제조는 다음의 3단계 절차로서 수행되었다: (1) 키토산 용액에 실리카 입자를 첨가시켜 필름을 형성시킨 후, (2) 이 필름을 알카리 용액에 침지시켜 실리카 입자를 제거하여 다공성의 키토산 막을 제조하였으며, (3) 다공성 키토산 막을 acetic anhydride를 사용하여 아세틸화시킴으로서 다공성 키틴 막을 제조하였다. 물리적 강도가 우수하고, 적절한 순수 투과량을 갖는 다공성 키토산 막과 키틴 막의 최적 제막조건이 제시되었다. 단백질 친화성을 부여하기 위해 다공성 키토산 막에 반응성 염료인 Cibacron Blue 3GA를 고정화시켰으며, BSA 단백질 및 lysozyme 효소의 흡착실험을 수행하여 친화 키토산 막 및 키틴 막의 단백질 결합용량을 측정하였다. 친화 키토산 막의 BSA 단백질 결합용량은 약 22 mg/mL이었으며, 친화 키틴 막의 lysozyme 효소 결합용량은 약 26 mg/mL로서 이는 키토산 또는 키틴을 기반으로 하여 제조된 hydrogel bead의 단백질 결합용량보다 수${\sim}$수십 배 큰 값으로서, 향후 막여과 크로마토그래피용 친화 막으로의 효과적인 활용이 기대된다.