• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.568-571
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• 1999

Aspergillus oryzae U1521, which was a genetically engineered strain, produced 1,000,600 U per g . glucose of extracellular alkaline protease within 72 h in a submerged fermentation. However, the alkaline protease was not detected during the first 24 h. Northern blot analysis indicated that the enzyme synthesis was repressed at the transcriptional level during the lag period. Both catabolite repression and pH of the growth medium significantly affected the enzyme production. Use of this enzyme as a silver recovery agent from used X-ray film was confirmed by experiments in the shake-flask scale.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.427-434
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• 1998

Xanthomonas sp. YL-37 이 생산하는 alkaline protease를 정제하기 위하여 ammonium sulfate로 침전시켜 회수하여 CM-cellulose ion exchange resin column에 주입한 후 Sephadex G-100 column에 2회 통과시켜 단일효소를 얻었으며 분자량은 약 62,000 dalton으로 단일 subunit로 되어있다. 정제효소의 반응최적온도는 5$0^{\circ}C$였으며 특히 2$0^{\circ}C$에서도 최적활성의 약 40%를 유지하였다. 금속염에 대한 영향은 MnSO$_4$, MgSO$_4$, CaCl$_2$,등에 의해서 효소활성이 촉진되었으나 HgCl$_2$, ZnSO$_4$, CuSO$_4$ 등에 의해서 효소활성이 저해되었다. 본 효소는 EDTA, EGTA에 의해서 강하게 저해를 받는 것으로 보아 metal ion을 갖고 있는 metaloenzyme으로 보이며 효소에 결합된 cofactor는 $Mn^{2+}$이었으며 정제한 alkaline protease의 NH$_2$-말단 아미노산은 alanine이었다. 본 효소의 Km값은 4.0 mg/$m\ell$이 었고 Vmax는 5,500 unit/$m\ell$이었다.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.450-455
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• 1998

Alkaline protease를 대량생산하는 Aspergillus oryzae를 만들기 위하여 A. oryzae의 alkaline pretense 유전자 alpA를 고발현시키는 plasmid pTAalp를 제조하고 이 plasmid로 A. oryzae M-2-3 균주를 형질전환시켰다. 16개의 형질전환체를 얻어 이들의 protease생산성을 skim milk 분해에 의한 halo 형성능에 의하여 확인하였다. 또한 protease 생산성이 증가한 형질전환체는 pTAalp가 multi-copy로 염색체 안에 integration되어 있음을 Southern blot에 의하여 확인하였고, 이들의 배양액을 polyacrylamide gel전기영동에 의하여 분석한 결과, 형질전환체 No. 14에서는 전체 분비단백질의 80-90%가 alkaline protease 임을 알 수 있었다. 간장원료 분해실험의 결과 No. 14에 의한 원료분해액은 간장 양조용 대조균에 의한 분해액보다 TN이 증가하였으며 원료분해율도 1.4-1.5배로 증가되었다.

• 미생물학회지
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• 제46권4호
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• pp.383-388
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• 2010

알칼리성 단백질 분해효소 고생산 돌연변이 균주 Vibrio metschnikovii L12-23, N4-8, KS1으로부터 알칼리성 단백질 분해효소를 암호화하는 vapK (Vibrio alkaline protease K) 유전자들을 PCR에 의하여 분리한 다음 DNA shuffling, error-prone PCR 방법과 같은 분자진화 기술을 통해 고활성 단백질 분해효소를 생산하는 재조합 V. metschnikovii 균주를 제작하였다. DNA shuffling 방법을 통해 변형시킨 vapK-1 유전자와 이 유전자를 주형으로 error-prone PCR 기법을 통해 재 변형된 vapK-2 유전자를 cloning한 후 V. metschnikovii KS1 균주에 역도입하여 재조합 균주를 제조하였다. 재조합 균주들의 단백질 분해 능력을 조사한 결과 vapK-2 유전자가 2 copy 도입된 재조합 균주의 경우 야생형 균주인 V. metschnikovii RH530에 비해 43.6배 높은 단백질 분해활성을 보였으며 숙주인 V. metschnikovii KS1에 비해 약 3.9배 향상된 단백질 분해 활성을 확인할 수 있었다. 변형된 vapK-1과 vapK-2 유전자를 야생형 vapK 유전자의 염기서열을 비교 분석한 결과 단백질 분해 능력의 활성에 영향을 미치는 active site를 제외한 부분에서 변화가 일어났음을 확인 할 수 있었다. 변형된 유전자 vapK-1을 two copy를 포함한 재조합 플라스미드를 가진 V. metschnikovii KS1을 30 L fermentor로 배양 하였을 때 배양 후 35 시간에 18,000 PU/ml의 활성을 보였으며, 이는 향후 산업용 균주로서 사용될 수 있는 가능성을 제시하였다.

• 미생물학회지
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• 제18권2호
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• pp.51-58
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• 1980

Mutational experiments were performed to improved to improve the protease productivity of Aspergillus flavus KU 153, which is selected among the wild strains. A UV-induced mutant strain having high protease productivity was obtained by the use of the clear zone method as a simple criterion for a primary screening test. Neutral and alkaline protease activities of hte mutant strain were higher than 1.8 times, comopared with those of the parental strain, respectively, while in the case of acid protease, it was 2.7 times. The mutant strain selected was more powerful in the production of cellulase and amylase, as well s protease in wheat bran, compared with those of the parental strain. protease production of the parental strain has reached maximum level at 3 days culture, while alkaline nad neutral protease production of the mutantstrain has reached at 2 days culture. On the other hand, the mutant strain formed the spore slowly, compared with the parental strain. Column chromatography of the neutral protease on DEAE-Sephadex A-50 showed that the mutant strain was not induced the formation of another neutral protease isozyme, but induced the variation in the function of regulatory gene.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.667-667
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• International Journal of Industrial Entomology and Biomaterials
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• 제33권2호
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• pp.78-84
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• 2016

The antioxidant activity of silkworm powder treated by proteolytic enzyme was investigated. Total protein content of silkworm power was assayed using BCA, Bradford assays and SDS-polyacrylamide gel electrophoresis (PAGE) with alkaline protease treatment conditions including temperature and pH. The optimum condition of alkaline protease treatment for silkworm powder was found to be $60^{\circ}C$ and pH 7. The alkaline protease treatment resulted in increased contents of free amino acids, total polyphenol and total flavonoid compared to control group. The silkworm hydrolysates showed excellent antioxidant activities in various in vitro models such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2 - azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS) radical scavenging activity. These results provide useful information for using silkworm powder as an ingredient in functional foods and for exploiting alkaline protease treatment to improve the extractability and bioactivity of a raw material.

• 한국식품위생안전성학회지
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• 제15권2호
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• pp.176-178
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• 2000

V. parahaemolyticus를 균주로 alkaline protease 생산을 위한 질소원의 종류, skim milk농도, NaCl농도, 금속이온의 종류를 조사하였다. 질소원인 peptone, tryptone, casamino acid, ski milk, phyton peptone중에서는 skim milk에서 활성이 월등히 높게 나타났고, skim milk의 농도에 따른 실험에서는 2%일 때 활성이 가장 높게 나타났다. NaCl의 농도에 따른 실험에서는 0%일 때 활성이 가장 높았고 농도가 높아질수록 활성이 낮아졌다. 금속이온에 따른 활성을 보면 금속이온이 들어가지 않았을 때 활성이 가장 높게 나타났고, 금속이온을 첨가하므로써 활성이 급격히 떨어졌다. 또한 CoCl$_2$, CuCl$_2$, HgCl가 첨가되었을 때는 활성이 전혀 나타나지 않았다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Applied Biological Chemistry
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• 제15권1호
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• pp.27-33
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• 1972

본(本) Monascus속(屬) 균주(菌株)를 Wheat bran 배지(培地)에 배양(培養)하여 여기서 얻은 Alkaline protease의 효소학적(酵素學的) 성질(性質)은 다음과 같다. 1) 본(本) Protease의 최적(最適) pH는 $10{\sim}12$에 위치(位置)하고 있으며 2) 최적온도(最適溫度)는 $50^{\circ}C$ 이었으며 3) 안정(安定) pH는 $5{\sim}12$에 위치(位置)하며 4) 본(本) Protease는 $40^{\circ}C$ 이하(以下)에서 안정(安定)하였으며 5) 금속(金屬) Ion 중(中) $Hg^#$와 $Fe^#$는 조해적(阻害的)으로 작용(作用)하여 $Pb^#$, $Ba^#$, $Co^#$, $Zn^#$, $Cu^#$등은 보호작용(保護作用)을 나타내었고 이 중(中) $Cu^#$는 $10^{-2}$ 농도(濃度)에서 본(本) protease의 실활(失活)을 강(强)하게 보호(保護)함을 알았다. 6) E.D.T.A는 본(本) Protease에 대(對)해서 아무 영향을 주지 않음을 알았다.