• Journal of Microbiology and Biotechnology
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• 제33권5호
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• pp.656-661
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• 2023

The aims of this study were to optimize the preparation of low-molecular-weight collagen using a proteolytic enzyme (alcalase) derived from the feet of Korean native chickens, and to characterize the process of collagen hydrolysis. Foreign bodies from chicken feet were removed using ultrasonication at 28 kHz with 1.36 kW for more than 25 min. The hydrolytic pattern and molecular weight distribution of enzyme-treated collagen from chicken feet were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography, respectively. Ideally, chicken feet should be treated at 100℃ for 8 h to obtain a high collagen content using hot water extraction. The collagen content of the chicken foot extract was 13.9 g/100 g, and the proportion of low-molecular-weight collagen increased with increasing proteolytic enzyme concentration and reaction time. When treated with 1% alcalase, the average molecular weight of collagen decreased rapidly to 4,929 Da within 5 h and thereafter decreased at a slower rate, reaching 4,916 Da after 7 h. Size exclusion chromatography revealed that low-molecular-weight collagen peptides of approximately 1,000-5,000 Da were obtained after hydrolysis with 1% alcalase for 1 h.

• 약학회지
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• 제37권2호
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• pp.129-135
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• 1993

Serratia sp. Y-4 was isolated from soil. Many characteristics of the strain and optimum cultivation condition for protease production were investigated.,The protease from Serratia sp. Y-4 was purified and studied for the properties of the enzyme. The isolated strain was identified to the genus Serratia. The strain was cultivated in 1%-casein, 0.5%-Na$_{3}$PO$_{4}$.7H$_{2}$O, 0.1%-NaCl, 0.05%-KCI, 0.02%-MgSO$_{4}$.7H$_{2}$O, 0.02%-CaCl$_{2}$.2H$_{2}$O, 0.02%-ZnSO$_{4}$.7H$_{2}$O, 0.02%-MnCl$_{2}$.4H$_{2}$O and 0.5%-soy bean oil at pH 7.0 for 35 hrs. The enzyme was purified about 5.89 fold from the culture broth with 31.1% recovery and 19,613 u/mg through ultrafiltration, ammonium sulfate, DEAE-sephacel and Superose-12 chromatography. The purified enzyme was identified as one band by isoelectric focusing, SDS- and native-PAGE. It has maxium activity at $37^{\circ}C$ and pH 9.0. Molecular weight of it is approx. 50 kD and pl is about 6.70. Its Km value for casein was 20 mg/ml. 5 mM-EDTA, 5mM-SDS, Ag$^{+1}$, Cu$^{+2}$, Hg$^{+2}$ and Pb$^{+2}$ inhibited the enzyme.

• 한국식품영양과학회지
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• 제18권3호
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• pp.348-355
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• 1989

Alkaline protease 생성능이 강한 Aspergillus fumigatus 균주를 토양에서 분리하고, 생성효소를 정제하여 특성을 조사한 결과 최적 pH는 9.0, pH안정성은 $pH\;8.0{\sim}10.0$, 최적온도는 $50^{\circ}C$였으며, $50^{\circ}C$이하의 온도에서 안정하나 그 이상의 온도에서는 급격한 효소 불활성화를 보였고, 금속염 $Mn^{++},\;Cu^{++},\;Ba^{++},\;Mg^{++}$ 등에 의해서 활성이 다소 증대되나 $K^+,\;Fe^{+++},\;Ag^{++},\;Pb^{++},\;Na^+,\;Ca^{++},\;Hg^+,\;Zn^{++}$에 의해 저해를 받았다. 활성저해제인 EDTA, 2,4-DNP, ${\varepsilon}-amino$ caproic acid에는 큰 저해를 받지 않으나, PCMB에 많은 저해를 받는 것으로 미루어 활성 부위가 SH기인 cystein protease로 추정되었다. Km값은 $8.33{\times}10^{-4}mole/{\ell}$, Vmax는 $47.62{\mu}g/min$였으며, casein과 hemoglobin을 trypsin보다 더 잘 분해하고, casein을 hemoglobin보다 잘 분해하였다.

• Journal of Applied Biological Chemistry
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• 제63권4호
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• pp.291-295
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• 2020

참기름 제조의 부산물인 참깨박의 활용도를 높이기 위하여 효소 처리에 의한 참깨박 불용성 단백질의 추출 조건을 조사하였다. 단백질 분해효소인 Alcalase, Flavourzyme, Neutrase, Protamex의 처리 결과를 대조구와 비교한 결과 Protamex가 고형분과 단백질 함량 증가에 효과적이었다. Protamex의 반응 조건(50 ℃, pH 6.0)에서 효소의 사용량은 탈지 참깨박의 1%, 효소반응시간은 3시간이 적당하였다. 효소 처리 효율을 향상시키기 위하여 참깨박을 열처리하면, 열처리 온도가 증가할수록 추출되는 단백질 함량은 증가하였으며 110이상에서는 미미하게 증가하였다. 세포벽 분해효소(Tunicase)와 단백질 분해효소의 병용처리가 단백질 가용화에 미치는 효과를 조사한 결과, 단백질 분해효소를 처리한 다음에 세포벽 분해효소를 처리하는 것이 가장 효과적이었다. 열처리(110 ℃, 10분) 후 Protamex와 Tunicase를 순차적인 처리로 단백질 함량이 열처리와 효소처리하지 않은 대조구의 약 3.6배(9.85→35.58 mg/mL), 열처리만 실시한 대조구의 약 2.2배(15.83→35.58 mg/mL) 증가하였다.

• 대한화장품학회지
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• 제26권1호
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• pp.81-92
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• 2000

Development of stabilized enzyme was attempted for cosmetic applications. Papain, a proteolytic enzyme, was stabilized through conjugation with a soluble carbohydrate biopolymer, SC-glucan$^{TM}$ . With a novel structure of the conjugation site, stability of the enzyme was significantly enhanced such that more than 90% of the initial activity retained after a month storage at 45$^{\circ}C$, while no activity were detected in native enzyme or enzyme simply mixed with SC-glucan$^{TM}$ after the storage. Conjugation with SC-glucan$^{TM}$ not only extended the half-life of the enzyme on storage at higher temperature, but was also found to protect enzymes against some components contained in cosmetic products for skin care. Cosmetic lotion containing 1 % papain conjugate was more effective and less irritative in exfoliating stratum corneum of human skin than the lotion containing 5% lactic acid, one of the current popular exfoliating agents.gents.

• 한국식품과학회지
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• 제5권3호
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• pp.174-177
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• 1973

한우(韓牛)의 사태근육(筋肉)에 단백질분해효소(蛋白質分解酵素)를 첨가(添加)해서 total tryptophan 과 free tryptophan을 spectrophotometric method로 분석(分析)한 결과(結果)는 다음과 같다. 1. 단백질분해효소(蛋白質分解酵素)를 0.01%, 0.05%, 0.1%로 첨가(添加)함에 따라 total tryptophan 과 free tryptophan은 계속 증가(增加)하였다. 2. Total tryptophan은 단백질분해효소(蛋白質分解酵素)를 육중량(肉重量)의 0.05% 첨가시(添加時) 그 증가율(增加率)이 가장 높았다. 3. 단백질분해효소(蛋白質分解酵素) 첨가(添加)에 따른 free tryptophan의 함량변화(含量變化)는 그다지 심하지 않았다.

• Journal of Microbiology
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• 제41권1호
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Preventive Nutrition and Food Science
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• 제14권4호
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• pp.335-342
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• 2009

Bioactive compounds were produced from carrot pomace by solid-state fermentation using Bacillus subtilis HA and Leuconostoc mesenteroides. The carrot pomace (CP) fermented by B. subtilis HA with 3% monosodium glutamate (MSG) showed higher production of various bioactive compounds, with 1.64 Pa·sn of consistency, 2.31% of mucilage content, 16.95 unit/g of fibrinolytic enzyme activity, 35.3 unit/g of proteolytic activity and 37.5 mg% of tyrosine content. The mucilage production was greatly dependent upon the concentration of MSG added. Most MSG added in CP was converted into mucilage (2.3%) including 0.83% poly-$gamma$-glutamic acid (PGA) with 1,505 kDa of molecular weight. The CP fermented secondly by Leuc. mesenteroides showed acidic pH and lower consistency. However, the fibrinolytic and proteolytic activities were increased. The secondly fermented CP showed the viable cell counts with $2.5{\time}108$ CFU/g of B. subtilis HA and $3.7{\time}109$ CFU/g of Leuc. mesenteroides, respectively. The freeze-dried fermented CP showed 2.88 Pa·sn of consistency, 24% of mucilage content and 104.9 unit/g of fibrinolytic enzyme activity, respectively. Also, the powder of fermented CP indicated viable cell counts of $8.0{\time}107$ CFU/g of B. subtilis and $4.0{\time}108$ CFU/g of Leuc. mesenteroides. Therefore, the fermented CP that was fortified with dietary fibers, fibrinolytic enzyme and probiotics could be utilized as valuable ingredients of functional foods in food or cosmetic industries.

• 한국식품영양과학회지
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• 제12권4호
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• pp.342-349
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• 1983

수산명산품(水産名産品)에 속(屬)하는 해삼내장(內臟)젓갈이 식품영양학적(食品營養學的)인 평가(評價) 및 그 가공(加工)에 관(關)한 기초자료(基礎資料)를 얻고자 본 연구를 착수하였으며, 해삼내장(內臟) 조직중(組織中)에 분포(分布)하는 단백질분해(蛋白質分解) 조효소(粗酵素)를 추출(抽出)하여 활성조건(活性條件)과 젓갈 숙성중(熟成中)의 유리(遊離)아미노산(酸) 및 단백질(蛋白質) 구성(構成) 아미노산(酸)의 조성변화(組成變化)에 관(關)하여 분석(分析) 검토(檢討)한 결과(結果)를 요약(要約)하면 다음과 같다. 1. 해삼내장(內臟) 조직중(組織中)에는 pH 3.1 $50^{\circ}C$, pH 5.7 $50^{\circ}C$ 그리고 pH 7.7 $45^{\circ}C$에 각각(各各) 최적활성조건(最適活性條件)을 갖는 세 종류(種類) 이상(以上)의 단백질분해효소(蛋白質分解酵素)의 존재(存在)가 확인(確認)되었다. 2. 이들 효소중(酵素中) pH 3.1 $50^{\circ}C$, pH 7.7 $45^{\circ}C$에 최적활성(最適活性)을 갖는 효소(酵素)들은 식염농도(食鹽濃度) 1%이상(以上)의 농도(濃度)에서 점차(漸次) 높은 조해(阻害)를 받았으며, pH 5.7 $50^{\circ}C$에 최적활성조건(最適活性條件)을 갖는 효소(酵素)는 식염농도(食鹽濃度) 1%에서 부활(賦活) 되었으며, 5%이상(以上)에서 조해(阻害)를 받았다. 3. 몇가지 염류(鹽類)이온에 의한 영향(影響)을 검토(檢討)한 결과(結果) pH 3.1 $50^{\circ}C$에서 최적조건(最適條件)을 보인 효소(酵素)는 실험(實驗)에 쓴 전(全) 염류(鹽類)이온에 의하여 조금씩 조해(阻害)를 받았으며, pH 5.7 $50^{\circ}C$에서 최적조건(最適條件)을 보인 효소(酵素)는 $Cu^{2+}$을 제외(除外)한 모든 염류(鹽類)이온에 의하여 부활(賦活)을 받았고, pH 7.7 $45^{\circ}C$에서 최적조건(最適條件)을 보인 효소(酵素)는 $Ca^{2+}$과 $Mn^{2+}$에 의하여서는 부활(賦活)을 받았으며, $Ba^{2+}$에 의하여서는 영향(影響)이 없었고 $Co^{2+}$, $Mg^{2+}$에 의하여서는 조해(阻害)를 받았다. 그리고 특(特)히 이 효소(酵素)들은 $Cu^{2+}$에 의하여서는 심(甚)한 조해(阻害)를 받았다. 4. 해삼내장(內臟) 조직(組織)에 분포(分布)하는 효소(酵素)들의 젓갈숙성일수(熟成日數)의 경과(經過)에 따른 활성(活性)에 미치는 영향(影響)을 검토(檢討)한 결과(結果), pH 3.1 $50^{\circ}C$와 pH 7.7 $45^{\circ}C$에서 활성최적조건(活性最適條件)을 보인 효소(酵素)들은 활성(活性)이 조금씩 약화(弱化)하여 갔으나, pH 5.7에서 활성최적조건(活性最適條件)을 보인 효소(酵素)는 숙성일수(熟成日數)의 경과(經過)에도 영향(影響)이 없었다. 5. 해삼내장(內臟)젓갈 숙성(熟成) 8일(日)째일 때는 생(生) 내장중(內臟中)의 단백질(蛋白質) 구성(構成)아미노산(酸)의 대부분(大部分)이 감소(減少)하였으며, 특(特)히 두드러지게 감소(減少)한 아미노산(酸)은 arginine, alanine, glutamic acid, glycine, serine, valine, threonine 및 lysine 등(等)이었고, methionine와 histidine 및 isoleucine 등(等)은 그 감소폭(減少幅)이 낮았다. 6. 유리(遊離)아미노산(酸) 조성(組成)을 분석(分析)한 결과(結果), 8일간(日間) 숙성(熟成)한 시료중(試料中)에는 glutamic acid, aspartic acid, leuine 및 lysine은 많은 량(量)이 함유(含有)되어 있었고, histidine, methionine, proline 및 tyiosine 등(等)은 그 함량(含量)이 낮았다. 그리고 젓갈 숙성과정중(熟成過程中)에는 대부분(大部分)의 유리(遊離)아미노산(酸)은 증가(增加)하였으며, 특(特)히 lysine, histidine, threonine, glutamic acid, methionine, valine, leucine 등(等)은 많은 증가(增加)를 보였다.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.469-474
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• 1999

Thermoactinomyces sp. E79 produced two types of thermostable alkaline proteases extracellularly. A minor protease was separated from a major protease by using DEAE-column chromatography. This enzyme was purified to homogeneity by ammonium sulfate and DEAE-Sepharose ion-exchange chromatography. The purified minor protease showed different biochemical properties compared to the major protease. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 36 kDa. Its optimum temperature and pH for proteolytic activity against Hammarsten casein were $70^{\circ}C$ and 9.0, respectively. The enzyme was stable up to$75^{\circ}C$ and in an alkaline pH range of 9.0-11.0. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF) and $Hg^{2+}, indicating that the enzyme may be a cysteine-dependent serine protease. In addition, the enzyme cleaved the endoproteinase substrate, succinyl-Ala-Ala-Pro-Phe-p- nitroanilide, and the $K_m$ value for the substrate was 1.2 mM.