• 제목/요약/키워드: Protease activity

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젓갈 성숙과정(成熟過程)에 있어 Protease 및 Formonitrogen의 변화(變化)에 관(關)한 연구(硏究)(1) (Changes in Protease and Formonitrogen of Salted Fish and Shellfish (1))

  • 서명자
    • Journal of Nutrition and Health
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    • 제6권2호
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    • pp.45-56
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    • 1973
  • Salted fish and shellfish have been widely used in Korea from olden times as side-dishes, although the processes and methods of pickling have varied depending on localities. The common raw materials for these subsidiary food articles include anchovy, shrimp, yellow corvina, oysters, octopus, top-shell, shellfish, pollack roe and pollack intestines. It must be pointed out here, however, that the salted stuffs now marketed locally are highly unscientific and unsanitary in the way they are processed and sold, and this has prompted this writer to undertake a study on these native food articles. The following findings have been obtained from this study on the changes in Formonitrogen and Protease Activity, effected by the density of salt and the degree of storing temperature, of the pickles of cedfish gills, codfish intestines, pollack intestines, shellfish, oysters, cuttle fish and octopus. 1) Codfish Gills The Protease Activity of the pickled codfish gills was greater in the groups of lesser doses of salt and higher degrees of storing temperature. The same was true in the case of Formonifrogen, too. The Formonitrogen of the pickled codfish gills was larger in the groups of lesser salt and higher teimperature. 2) Codfish Intestines The amount of Formonitrogen of the pickled codfiah intestines became greater, as time went by, in the groups of lesser salt than those of larger doses of salt, with the speed of its formation getting faster as the storing temperature rose from $5^{\circ}C$ to $15^{\circ}C$ and $30^{\circ}C$. The Protease Activity was also greater in the groups of lesser salt and higher temperature. The group, stored at 10% salt and $30^{\circ}C$, rotted in five days. 3) Pollack Intestines The amount of Formonitrogen of the pickled pollack intestines was greater in the groups of lesser amount of salt and higher degrees of storing temperature. The Protease Activity of the pickled pollack intestines began decreasing from the 11th day after the pickling in the groups stored at colmparatively high degrees of temperature$(15^{\circ}C-30^{\circ}C)$, while that of the group stored at $5^{\circ}C$ kept rising. The effects of the amount of salt were little. The group stored at 15% salt and $30^{\circ}C$ started rotting on the 13th day while that stored at $30^{\circ}C$ decayed on the 7th day. The group stored at 20% salt and $30^{\circ}C$ rotted on the 9th day. 4) Oysters The amount of Formonitrogen of the pickled oysters became greater as the storing temperature rose and the doses of salt were lowered. The Protease Activity was not affected at any measurable degree by the density of salt in the group stored at $5^{\circ}C$, but became greater as the storing temperature rose to $15^{\circ}C$ and $30^{\circ}C$. The group stored at 10% salt and $30^{\circ}C$ rotted on the 5th day while that stored at 20% salt and $30^{\circ}C$ on the 13th day. 5) Shellfish The amount of Formonitrogen of the pickled shellfish became greater, as time went by, it the groups of lower consistency of salt than the groups of higher density of salt, although the decay of the former groups was faster than the latter groups. The density of salt best fitted for the pickling appeared to be about 20% with the storing temperature to be $15^{\circ}C$, at which the pickled stuff became most tasty on the 7th day. The oysters stored in three groups at $5^{\circ}C$, $15^{\circ}C$ and $30^{\circ}C$ respectively showed the greatest Protease Activity alike at 0% of salt, but the activity declined as the density of salt increased. The Protease Activity of each group rose for the first 11 days after the pickling, but began declining from the 13th day onward, with the groups of higher temperature retaining higher Protease Activity than the groups of lower temperature. 6) Cuttlefish Both the amount of Formonitrogen and the degree of Protease Activity of the pickled cuttlefish were greater in the groups of lower density of salt and higher degree of storing temperature. The oysters pickled at 10% salt and $15^{\circ}C$ degenerated on the 13th day while that of 10% salt and $30^{\circ}C$ deteriorated on the 7th day. 7) Octopus Both the Formonitrogen and the Protease Activity of the pickled octopus were greater in the groups of lower density of salt, but as time went by, the Protease Activity in all groups dwindled after a climbing. In general, the Formonitrogen and the Protease Activity of the pickled oysters became greater as the storing temperature got higher. One group stored at 10% salt and $15^{\circ}C$ rotted in 13days while another group stored at $30^{\circ}C$ decayed in 7 days.

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E. coli에서 발현된 human HtrA1 단백질의 정제와 HtrA1의 serine protease 활성 조건에 관한 연구 (Purification of Human HtrA1 Expressed in E. coli and Characterization of Its Serine Protease Activity)

  • 김경희;김상수;김구영;임향숙
    • 생명과학회지
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    • 제16권7호
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    • pp.1133-1140
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    • 2006
  • E. coli HtrA (High temperature requirement protein A)의 human homologue 중 하나인 HtrA1은 IGFBP를 절단하여 IGF의 활동을 조절하는 serine protease으로 알려졌다. HtrA1의 serine protease 활성이 여러 질병의 발병 mechanism과 연관성을 가진 것으로 예상되고 있지만, 이런 상관관계를 밝히기 위해서 기본적으로 필요한 다량의 HtrA1 단백질의 발현 및 정제조건과 HtrA1 serine protease의 최적 활성조건이 확립되어 있지 않은 상황이다. 따라서 본 연구에서는 pGEX 시스템을 이용하여 E. coli에서 mature HtrA1인 ${\Delta}149(WT)$와 catalytic site mutant인 ${\Delta}149(S328A)$를 85%의 순도로 1 liter 배양 시, 정제된 단백질을 각각 $400{\mu}g,\;520{\mu}g$ 얻을 수 있는 발현조건을 정립하였다. 또한 HtrA1 serine protease 활성은 protease의 농도와 substrate와의 반응시간에 dependent하며, substrate와의 반응온도가 $42^{\circ}C$일 때 최적의 serine protease활성을 나타내는 것을 알 수 있었다. 특히 $200{\mu}M$의 HtrA1 serine protease를 $37^{\circ}C$에서 3시간 반응 시켰을 때, substrate로 사용한 ${\beta}-casein$의 약 50%가 절단되는 것을 관찰하였다. 따라서 이 반응조건에 사용한 HtrA1의 양을 1 unit으로 하여 HtrA1의 serine protease활성을 여러 조건에서 비교 분석할 수 있다 본 연구에서 정립한 mature HtrA1을 다량으로 얻을 수 있는 발헌 및 정제조건과 serine protease 최적 활성조건은 HtrA1의 serine protease 활성과 생물학적 기능의 상관관계를 이해하는데 활용될 수 있을 것이다.

Aspergillus fumigatus이 생산하는 Alkaline protease의 특성과 작용양상 (Characteristics and Action Pattern of Alkaline Protease produced from Aspergillus fumigatus)

  • 차원섭;최청
    • 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.

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오징어 내장에서 분리한 Protease 특성의 모니터링 (Monitoring Characteristics of Protease Isolated from Squid Viscera)

  • 서지형;정용진;이기동;이명희
    • 동아시아식생활학회지
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    • 제9권2호
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    • pp.195-199
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    • 1999
  • 오징어 내장에서 분리한 protease를 중심합성 계획법을 이용해서 제한조건을 동일하게 하고 pH와 온도를 변수로 protease활성을 모니터링 하였다. 그 결과 protease활성은 41.75$^{\circ}C$, pH 6.02에서 78.65unit로 최고치를 나타내었으며, 이때 $R^2$는 0.8461로서 10% 이내에서 유의성이 인정되었다. 또한 오징어 내장 protease는 50mM의 $Na^{+}$ 첨가에서 활성이 저해되었으나 $Mg_2$$^{+}$에 의해서는 상승효과를 보였으며 km 값은 0.12mM이었다.

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Characterization and Production of Thermostable and Acid-stable Extracellular Fibrinolytic Enzymes from Cordyceps militaris

  • Kim, Seon-Ah;Son, Hong-Joo;Kim, Keun-Ki;Park, Hyun-Chul;Lee, Sang-Mong;Cho, Byung-Wook;Kim, Yong-Gyun
    • International Journal of Industrial Entomology and Biomaterials
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    • 제22권2호
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    • pp.83-93
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    • 2011
  • Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity

  • Li, Xiaoguang;Zhang, Qian;Gan, Longzhan;Jiang, Guangyang;Tian, Yongqiang;Shi, Bi
    • Journal of Microbiology and Biotechnology
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    • 제32권1호
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    • pp.99-109
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    • 2022
  • This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80℃) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70℃ and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Alkaline Protease Production from Bacillus gibsonii 6BS15-4 Using Dairy Effluent and Its Characterization as a Laundry Detergent Additive

  • Polson Mahakhan;Patapee Apiso;Kannika Srisunthorn;Kanit Vichitphan;Sukanda Vichitphan;Sukrita Punyauppa-path;Jutaporn Sawaengkaew
    • Journal of Microbiology and Biotechnology
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    • 제33권2호
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    • pp.195-202
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    • 2023
  • Protease is a widely used enzyme particularly in the detergent industry. In this research, we aimed to isolate alkaline protease-producing bacteria for characterization as a laundry detergent additive. The screening of alkaline protease production was investigated on basal medium agar plus 1% skim milk at pH 11, with incubation at 30℃. The highest alkaline protease-producing bacterium was 6BS15-4 strain, identified as Bacillus gibsonii by 16S rRNA gene sequencing. While the optimum pH was 12.0, the strain was stable at pH range 7.0-12.0 when incubated at 45℃ for 60 min. The alkaline protease produced by B. gibsonii 6BS15-4 using dairy effluent was characterized. The optimum temperature was 60℃ and the enzyme was stable at 55℃ when incubated at pH 11.0 for 60 min. Metal ions K+, Mg2+, Cu2+, Na+, and Zn2+ exhibited a slightly stimulatory effect on enzyme activity. The enzyme retained over 80% of its activity in the presence of Ca2+, Ba2+, and Mn2+. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit the enzyme activity, whereas phenylmethylsulfonyl fluoride significantly inhibited the protease activity. The alkaline protease from B. gibsonii 6BS15-4 demonstrated efficiency in blood stain removal and could therefore be used as a detergent additive, with potential for various other industrial applications.

Enzymatic Activities of Allergen Extracts from Three Species of Dust Mites and Cockroaches Commonly Found in Korean Home

  • Jeong, Kyoung-Yong;Kim, Chung-Ryul;Yong, Tai-Soon
    • Parasites, Hosts and Diseases
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    • 제48권2호
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    • pp.151-155
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    • 2010
  • Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.

Octopus vulgaris의 장관으로부터 분리한 단백질 분해효소 생성 균주와 생성된 효소의 특성 (Protease Properties of Protease-Producing Bacteria Isolated from the Digestive Tract of Octopus vulgaris)

  • 류청;;;양지영
    • 생명과학회지
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    • 제23권12호
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    • pp.1486-1494
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    • 2013
  • Octopus vulgaris의 장관으로부터 단백질 가수분해력과 활성을 측정함으로서 높은 단백질분해효소 생성능을 가진 균을 분리하여 동정하였다. 균이 생성한 단백질분해효소는 황산암모늄침전, cellulose CM-52 양이온 교환 크로마토그래피, DEAE-Sephadex A50 음이온 교환 크로마토그래피의 3단계를 통해 정제하였다. 장관으로부터 분리한 균중 가장 높은 단백질분해효소 생성능을 가진 균은 Bacillus sp. QDV-3로 나타났으며 이균을 분리한 후 표현형 분석, 생화학적 특성, 16S rRNA 유전자염기서열분석을 통해 Bacteria역, Firmicutes문, Bacilli강, Bacillales목, Bacillaceae과, Bacillus속으로 Bacillus flexus와 99.2%의 유사성을 보이는 것으로 확인하였다. 균이 생성한 단백질 분해효소를 QDV-E로 지정하였으며 61.6 kDa의 분자량을 나타내었다. 이 효소는 pH 9.0~9.5에서 활성을 나타내었고 최적온도는 $40^{\circ}C$였으며 $50^{\circ}C$에서는 60분간 96% 이상의 활성을 보유하였다. Phenyl methyl sulfonyl fluoride (PMSF)에 의하여 활성이 억제 되었으므로 세린 알칼리성 단백 분해 효소인 것으로 결론지었다. 금속이온인 $Mn^{2+}$$Mg^{2+}$에 의하여 효소활성 상승효과를 보였으며 $Ba^{2+}$, $Zn^{2+}$, 그리고 $Cu^{2+}$에 의하여 활성이 억제되었다.

재래식 메주로부터 분리한 Mucor racemosus f. racemosus PDA 103이 생산하는 Fungal Protease 특성 (Characteristics of Fungal Protease Produced by Mucor racemosus f. racemosus from Korean Traditional Meju)

  • 임성일;유진영
    • 한국미생물·생명공학회지
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    • 제27권6호
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    • pp.466-470
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    • 1999
  • Protease production and its characteristics were investigated with Mucor racemosus f. racemosus PDA 103 which was isolated from Korean traditional meju. Optimum culture conditions of the strain for the production of the protease in basic medium[bean(Baektae):H2O=1:1(w/v)] were as follows: pH 6, 3$0^{\circ}C$ and 72hrs. Optimum pH and temperature for the enzyme activity of the protease produced by Mucor racemosus f. racemosus were pH 5 and 5$0^{\circ}C$, respectively. The enzyme was relatively stable a pH2.0~5.0 and at temperature below 4$0^{\circ}C$. Phenylmethane-sulfonyl fluoride and Ag+ inhibited the enzyme activity. This indicates that the enzyme is serine protease. Km value was 0.9$\times$10-4M and Vmax value was 5.93$\mu\textrm{g}$/min. This enzyme hydrolyzed casein more rapidly than bovine albumin.

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