• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.430-434
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• 2004

Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-$\beta$-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1291-1300
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• 2018

Objective: This experiment was conducted to investigate the effects of dietary crude protein (CP) level and exogenous protease supplementation on growth performance, serum metabolites, carcass traits, small intestinal morphology and endogenous protease activity in broiler chickens reared under a tropical climate. Methods: A total of 480 day-old male broiler chicks were randomly assigned to eight dietary treatments in a $4{\times}2$ factorial arrangement. The main effects were CP level (21.0%, 19.7%, 18.5%, or 17.2% from 1 to 21 days and 19.0%, 17.9%, 16.7%, or 15.6% from 22 to 35 days) and protease enzyme supplementation (0 ppm or 500 ppm). All experimental diets were fortified with synthetic feed-grade lysine, methionine, threonine and tryptophan to provide the minimum amino acid recommended levels for Cobb 500. Results: Reducing dietary CP linearly reduced (p<0.05) growth performance, serum albumin, total protein, and carcass traits and increased (p<0.05) serum triglycerides and abdominal fat. There was no consistent effect of reducing dietary CP on morphological parameters of the intestine and on the pancreatic and intestinal endogenous protease activity (p>0.05). Protease supplementation improved (p<0.05) feed conversion ratio, body weight gain, carcass yield and intestinal absorptive surface area. Conclusion: Protease supplementation, as measured by growth performance, intestinal morphology and carcass yield, may alleviate the detrimental effects of low protein diets in broiler chickens.

• 한국식품영양과학회지
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• 제42권9호
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• pp.1461-1466
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• 2013

본 연구에서는 과일(무화과, 키위, 파인애플)에서 추출한 단백질분해 조효소와 전통발효제(개량누룩, 전통누룩, 메주, 입국)에서 추출한 단백질분해 조효소의 특징을 비교하였다. 전통발효제에서 추출한 단백질분해 조효소는 과일에서 추출한 단백질분해조효소보다 높은 온도($70^{\circ}C$), 높은 염 내성(1~3%), 그리고 낮은 pH(3~6)에서 활성을 보였다. 같은 조건에서 활성을 비교하였을 때, 바이오 누룩>입국>전통누룩>무화과>파인애플>메주>키위 순으로 활성이 높았다. 이러한 결과는 과일에서 추출한 단백질분해 조효소보다 전통발효제에서 추출한 단백질분해 조효소의 활성이 우수하고 고온, 산성, 그리고 염의 조건에서도 활성이 저하되지 않으므로, 소시지나 치즈 제조 등의 다양한 식품산업에 이용될 수 있는 가능성을 보여준다.

• Applied Biological Chemistry
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• 제38권6호
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• pp.534-540
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• 1995

토양에서 분리된 호알칼리성 coryneform bacteria TU-19는 적어도 세종류의 단백질분해효소(Protease I, II, III)를 생성분비하였다. 이 균주의 효소생산과 관련된 배양조건을 조사해본 결과 최적온도 및 pH는 각각 $30^{\circ}C$와 10.0인 것으로 나타났다. 이틀 배양된 배양액으로부터 이 효소들을 정제하기 위해서 ammonium sulfate fractionation, gel filtration 및 QAE-Sephadex column chromatography 등을 순차적으로 행하였다. 그 결과 이들 세종류의 효소는 SDS-PAGE pattern으로 평가했을 때 single band로 순수 정제되었으며, 각각의 분자량은 120, 80, 45 kDa이었다. 정제된 효소의 특성을 살펴보면 Pretense I과 II의 최적 pH와 온도는 각각 $10.5,\;45^{\circ}C$이었으며, Protease III는 11.0과 $50^{\circ}C$로 나타났다. 또한 이들 세효소는 10 mM PMSF 농도에서 효소활성을 완전히 상실하였으며, pCMB, 1,10-phenanthroline, IAA, EDTA에는 별 영향이 없는 것으로 보아 serine protease의 일종으로 생각된다.

• 한국식품영양학회지
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• 제8권3호
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• pp.192-198
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• 1995

Cheese milk를 기질로 하여 pronase 등 protease와 palanatase ML 등 lipase를 이용하여 EMC를 제조하였다. Cheese milk 가수분해에 관여하는 각 protease의 반응 조건을 살펴본 결과, pronase-3$0^{\circ}C$, pH 7.0, PANCREATIO-4$0^{\circ}C$, pH 8.0, pacific protease-3$0^{\circ}C$, pH 7.0과 Asp. Sp. 기원 protease-5$0^{\circ}C$, pH 8.0에서 최대의 활성을 보였으며, lipase의 반응 조건은 pancreatic lipase-5$0^{\circ}C$, pH 8.0, palatase ML-5$0^{\circ}C$, pH 7.0과 Candida cylindracea 기원 lipase-4$0^{\circ}C$, pH 6.0 이었다. 최적 반응 조건에서 가수분해를 행한 결과 반응 시간이 증가할수록 유리아미노산의 양과 유리지방산의 양이 증가하는 경향을 보였다. Pacific protease와 pronase에 의해 가수분해시 0.67mg/ml과 0.74 mg/ml의 유리 아미노산을 생성하였으며, 유리 아미노산 중 glutamic acid와 leucine이 가장 많은 함량을 보였다. Candida cylidracea 기원 lipase를 이용하여 가수분해시 가장 많은 유리 지방산 생성량을 보였다. 각 EMC의 지방산 조성을 살펴본 결과 butyric acid, palmitic acid, stearic acid와 oleic acid의 함량이 보였다. Pancreatin에 의해 가수분해 후 각 lipase를 사용하여 제조한 EMC의 관능검사 결과, pancreatic lipase를 이용한 EMC가 가장 쓴맛이 강하였고, palatase에 의한 EMC는 가장 바람직한 cheese 향기를 가지고 있다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권7호
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• pp.998-1003
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• 2016

Although exogenous protease enzymes have been used in poultry diets quite extensively, this has not been the case for pig diets. In general, due to their better gut fermentative capacity and longer transit time, pigs have greater capacity to digest dietary proteins than poultry. However, in early-weaned piglets, the stress brought about by weaning adversely affects the digestion of dietary proteins. Therefore, a study was conducted to determine the effects of a commercial protease enzyme in weanling pigs. Indices of growth, nutrient digestibility, blood profiles, fecal microflora, fecal gas emission and fecal scores were measured during the study. A total of 50 weanling pigs ($6.42{\pm}0.12kg$) at 28 d of age were randomly assigned to receive 1 of 2 dietary treatments: i) control diet (corn-soy based) with no supplemental protease (CON), and ii) control diet+200 g/ton protease (PROT) for 42 d. A completely randomized design consisting of 2 treatments, 5 replicates, and 5 pigs in each replicate was used. Growth performance in terms of body weight ($27.04{\pm}0.38kg$ vs $25.75{\pm}0.39kg$; p<0.05) and average daily gain ($491{\pm}7.40g$ vs $460{\pm}7.46g$; p<0.05) in PROT fed pigs were increased significantly, but gain per feed ($0.700{\pm}0.01$ vs $0.678{\pm}0.01$; p>0.05) was similar between treatments at d 42. Relative to CON pigs, PROT fed pigs had increased (p<0.05) apparent total tract digestibility ($84.66%{\pm}0.65%$ vs $81.21%{\pm}1.13%$ dry matter and $84.02%{\pm}0.52%$ vs $80.47%{\pm}1.22%$ nitrogen) and decreased (p<0.05) $NH_3$ emission ($2.0{\pm}0.16ppm$ vs $1.2{\pm}0.12ppm$) in the feces at d 42. Except for a decreased (p<0.05) in blood creatinine level, no differences were observed in red blood cell, white blood cell, lymphocyte, urea nitrogen, and IgG concentrations between treatments. Fecal score and fecal microflora (Lactobacillus and E. coli) were also similar between CON and PROT groups. Overall, the supplementation of protease enzyme in weanling pigs resulted in improved growth rate and nutrient digestibility. Exogenous protease enzyme reduced fecal $NH_3$ emission, thus, potentially serving as a tool in lowering noxious gas contribution of livestock production in the environment.

• Applied Biological Chemistry
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• 제41권1호
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• pp.6-12
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• 1998

폐단백질을 활용하는 방도의 하나로 폐단백질 자원으로 부터 불용성 단백질의 분리 효율성을 높이고 기능성을 개선하기 위하여 protease를 생산하는 Aspergillus sp. MS-18 균주를 토양으로 부터 분리하고 이 균주가 생산하는 효소를 정제하여 특성을 살펴보았다. 효소 생산을 위한 최적 배양조건은 3% arabinose, 0.5% polypepton, 0.1% ammonium sulfate, 0.1% magnesium chloride 첨가로 3 일 배양이었다. 효소는 ion exchange chromatography, gel filtration 등으로 16.9 배 정제할 수 있었으며 비활성역가는 340.4 unit/mg이었다. 정제효소는 polyacryl amide gel 전기영동상 단일 밴드로 나타났으며, 분자량은 30,000 정도로 추정되었고 결정구조는 모서리가 둥그스럼한 막대 모양이었다. 정제 효소의 최적작용 pH와 온도는 9.0, $60^{\circ}C$였으며, pH 7.0-12.0까지 $50^{\circ}C$에서 안정하였다. 금속이온중 $Na^+$, $Mg^{2+}$, $Mn^{2+}$등에 의해 활성이 증대 되었으나, $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$에 의해 효소 활성이 저해되었고 저해제중 ethylenediaminetetra acetic acid와 phenyl methanesulfonyl fluoride에 의한 활성 저해가 관찰되어 금속 이온이 효소 활성에 관여하는 serine protease로 추정되었으며 정제효소의 Km, Vmax는 $29.33\;{\mu}mole/L$, $5.13\;{\mu}g/min$이었다.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.56-61
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• 1997

An alkaline protease producing microorganism was isolated from korean traditional Meju and identified as Scopulariopsis brevicaulis. The optimum culture condition of Scopulariopsis brevicaulis for the production of alkaline protease was as follow: 2% soluble starch, 0.2$, tryptophan, 0.1% (NH$_{4}$) $_{2}$S$_{2}$O$_{8}$ 0.2% NaHPO$_{4}$, pH 7.5, 35$\CIRC $C. The optimum pH and temperature for the enzyme activity of alkaline protease producing Scopulariopsis brevicaulis were pH 9.0 and 50$\circ $C, respectively. The enzyme was relatively stable at pH 6.0~11.0 and at temperature below 40$\circ $C. The activity of the enzyme was inhibited by Hg$^{2+}$ whereas Cu$^{2+}$ gave rather activating effects on the enzyme activity. Phenylmethanesulfonyl fluoride inhibited the enzyme activity. This result indicates that serine is very important role in this enzyme. Km value for casein was 1.2410$^{4}$ M/L, V$_{max}$ value for casein was 25.99 $\mu $g/min. This enzyme hydrolyzed casein more rapidly than the hemoglobin.

• 한국미생물·생명공학회지
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• 제19권4호
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• pp.383-389
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• 1991

저온에서 높은 활성을 나타내는 알칼리성 protease를 생산하기 위하여 여러가지 시료로부터 집적배양에 의해 저온성 세균을 분리하였다. 분리된 세균은 저온.알칼리성 Pseudomonas sp.인 것으로 판명되었으며, 효소생산을 위한 균생육의 최적 pH는 10.0, 온도 $20^{\circ}C$에서 4일간 배양하였을 때였다. 이 효소 활성의 최적 pH 및 온도는 각각 pH 10.5 및 $40^{\circ}C$였으며, pH 및 열안정성은 각각 pH 7.0~13.0, 온도 $50^{\circ}C$이하의 범위에서 비교적 안정하였다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.330-336
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• 2004

Fifty-two pathogenic Vibrio parahaemolyticus strains were isolated from the environments of Busan and Yeosu, Korea. Forty-three of these strains showed protease activities, whereas 4 strains showed $\alpha / \beta$ hemolysin activities and 6 strains had urease activities. Their pathogenic factors were not overlapping except one strain, which had both protease and hemolysin activities. The 6 urease-positive strains (V. parahaemolyticus YKB4, YKB14, S25, YFB20, YFO21, and YFO22) showed the same biochemical characteristics as a reference strain [V. parahaemolyticus KCTC 2471 (urease-negative)], except for urease production. The 6 urease-positive strains showed different urease activities in their culture supernatant during the growth. The urease activity of S25 increased sharply at the late exponential phase, and was the highest at the initial stationary phase and was kept until the late stationary phase. The other 5 isolates, except C25, showed urease activities at the mid-stationary phase and increased steadily until the late stationary phase, when the urease activity was maximal. To compare the degree of virulence of V. parahaemolyticus with different pathogenic factors, hemolysin, protease, or urease-positive strains were injected into groups of 10 each of ICR mice (7- to l0-week-old males). The lethal rates of urease-positive V. parahaemolyticus, YKB14, YKB4, and S25, were significantly high, being 50, 70, and 80%, respectively. Protease-positive V. parahaemolyticus strains FM39 and FM50 showed 40% and 60% of lethal rate, respectively. Hemolysin-positive V. parahaemolyticus strains S34 and S72 had no mortality, similar to nonpathogenic V. parahaemolyticus FM12.