• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.89-92
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• 2001

Aspergillus niger has been used as a host system to express many heterologous proteins. It has various advantages over other expression systems in that it is a small eukaryotic GRAS (Generally Recognized aS Safe) organism with a capacity of secreting large amount of foreign proteins. However, it has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The proteases deficient mutants of A. niger were obtained using UV -mutagenesis. A total of 1 ${\times}$ $10^5$ spores were irradiated with 10-20% survival dose of UV, 600J/M2 at 280nm, and the resulting spores were screened on the casein -gelatin plates. Ten putative protease deficient mutants were further analyzed on the starch plates to differentiate the pro from the secretory mutant. An endogenous extracellular enzyme, glucose oxidase, was also examined to confirm that the mutant phenotype was due to the proteases deficiency rather than the mutation in the secretory pathway. The reduced proteolytic activity was measured using SDS-fibrin zymography gel, casein degradation assay, and bio-activity of a supplemented hGM -CSF (human Granulocyte-Macrophage Colony Stimulating Factor). Comparing with the wild type strain, less than 30 % of proteolytic activity was observed in the culture filtrate of the protease deficient mutant (pro -20) without any notable changes in cell growth and secretion.

• BMB Reports
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• 제28권2호
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• pp.138-142
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• 1995

An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a $K_m$ value of 1.1 ${\mu}M$ at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.

• 한국식품위생안전성학회지
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• 제15권2호
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• pp.173-175
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• 2000

V. parahaemolyticus 종류들이 gelatin분해 확인 실험에서 큰 clear zone을 형성했고, V. alginolyticus 분리균주도 V. parahaemolyticus와 비슷한 clear zone을 형성했다. V. fluvialis KCTC 2473은 clear zone을 형성하지 못했다. Hemolytic activity측정 실험에서는 V. parahaemolyticus 분리균주(2)가 가장 활성이 크게 나타났고, V. parahaemolyticus ATCC 17802, V. parahaemolyticus 분리균주(1)과 V. mimicus ATCC 33653도 높은 활성을 나타냈으며, 나머지 다른 비브리오균은 활성이 나타나지 않았다. Azocasein분해 활성 실험에서는 V. parahaemolyticus ATCC 17802, 분리균주(1), 분리균주(2)는 200EU/m1전후로 활성이 높게 나타났고, 나머지는 110EU/ml전후로 활성이 상대적으로 낮게 나타났다.

• 한국축산식품학회지
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• 제43권1호
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• pp.46-60
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• 2023

Slaughterhouse blood is a by-product of animal slaughter that can be a good source of animal protein. This research purposed to examine the functional qualities of the blood plasma from Hanwoo cattle, black goat, and their hydrolysates. Part of the plasma was hydrolyzed with proteolytic enzymes (Bacillus protease, papain, thermolysin, elastase, and α-chymotrypsin) to yield bioactive peptides under optimum conditions. The levels of hydrolysates were evaluated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The antioxidant, metal-chelating, and angiotensin I-converting enzyme (ACE) inhibitory properties of intact blood plasma and selected hydrolysates were investigated. Accordingly, two plasma hydrolysates by protease (pH 6.5/55℃/3 h) and thermolysin (pH 7.5/37℃/3-6 h) were selected for analysis of their functional properties. In the oil model system, only goat blood plasma had lower levels of thiobarbituric acid reactive substances than the control. The diphenyl picrylhydrazyl radical scavenging activity was higher in cattle and goat plasma than in proteolytic hydrolysates. Ironchelating activities increased after proteolytic degradation except for protease-treated cattle blood. Copper-chelating activity was excellent in all test samples except for the original bovine plasma. As for ACE inhibition, only non-hydrolyzed goat plasma and its hydrolysates by thermolysin showed ACE inhibitory activity (9.86±5.03% and 21.77±3.74%). In conclusion, goat plasma without hydrolyzation and its hydrolysates can be a good source of bioactive compounds with functional characteristics, whereas cattle plasma has a relatively low value. Further studies on the molecular structure of these compounds are needed with more suitable enzyme combinations.

• 생명과학회지
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• 제11권1호
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• pp.76-82
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• 2001

Fifteen species of microorganisms were isolate form the intestines of fishes, fish feed, and ferment. Eleven microorganisms except HY4, HY8, HY12, and HY13 were Gram-positive, and HY1, HY2, HY3, HY5, HY6, and HY7 produced lactic acid. The species of HY1, HY2, HY3, HY4, HY5, HY6, HY13, and HY14 showed some growth in the medium containing 1% of NaCl. Except HY6, HY7, HY8, HY12 and HY5, 10 isolates had proteolytic activity, whereas only HY13 and HY14 had lipase activity. From all the results four isolates (HY3, HY4, HY13 and HY14) were chosen for the degradation of fish wastes. There was no mutual inhibition among the microorganisms, and the optimum temperature and pH for the growth of the mixed culture were found to be 3 2$\^{C}$ and 7, respectively. Under the optimum growth conditions the maximum optical density and the maximum specific growth rate were estimated to be 2.35 and {TEX}$0.46h^{-1}${/TEX}, respectively. Major microorganisms in the mixed culture at the log-phase were HY3 and HY4, which occupied 70%. The degrading efficiency of fish waste by the mixed microorganisms was 2.3 times higher, compared to control. The total amount of free amino acids in the degraded products from fish wastes was 39g/100g protein and little odor was produced by the mixed microorganisms after 48 hours.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2006년도 Spring Conference
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• pp.77-90
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• 2006

Beta-amyloid peptide (A$\beta$) is a major component of senile plaques and its aggregation is considered to play a critical role in pathogenesis of Alzheimer's disease (AD). Aggregation of A$\beta$ could result from both increased synthesis and decreased degradation of A$\beta$. Our laboratory is interested in understanding the mechanism of A$\beta$ degradation in brain. Recently our laboratory identified a bacterial gene (SKAP) from Streptomyces sp KK565 whose protein product has an activity to cleave A$\beta$ and thus reduce the A$\beta$-induced neurotoxicity. The sequence analysis showed that this gene was closely related to aminopeptidase. Maldi-Tof analysis showed that the recombinant SKAP protein expressed in E. coli cleaves both A$\beta$ 40 and A$\beta$ 42 at the N-terminal of A$\beta$ while an aminopeptidase from Streptomyces griseus (SGAP) cleaves at the C-terminal. We also identified a mammalian homolog of SKAP and the recombinant mammalian protein expressed in Sf-9 insect cells showed a similar proteolytic activity to SGAP, cutting A$\beta$ at the C-terminus. I well discuss the detailed mechanism of the enzyme action and its functional implication in AD.

• 농업과학연구
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• 제49권4호
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• pp.1047-1053
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• 2022

The purpose of this study was to compare the effect of solid-state fermentation on soybean using three microbial strains under four different fermentation times. Soybean was fermented for 12, 24, 36 or 48 hours with highly proteolytic microbes, either Bacillus amyloliquefaciens (BA), B. subtilis (BS), or B. subtilis var. natto (BN), and levels of total protein concentration, protein distribution, and antioxidant activity were analyzed. Total protein was highest in the BS 12 h group (9.21 ㎍·µL^-1) and lowest in BN 48 h (6.80 ㎍·µL^-1), respectively (p < 0.001). Furthermore, three microbes decomposed large molecular weight proteins as well as major allergens of soybean such as β-conglycinin, Gly m Bd 30K, and glycinin. Each treatment group showed the highest degradation rate at 48 h fermentation and among the three microbes, BS showed a relatively higher degradation rate. The radical scavenging ability, known as an indicator of antioxidant activity, showed a significant increase in all treatment groups except BA 24 h. The results from this study suggest that protein concentration, and degradation and antioxidant activity were affected by different types of microbial trains and fermentation period and that B. subtilis fermentation might be the most effective way to increase nutritional and functional properties of soybean.

• Biomolecules & Therapeutics
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• 제15권1호
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• pp.16-26
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• 2007

Tissue plasminogen activator (tPA) is a serine protease catalyzing the proteolytic conversion of plasminogen into plasmin, which is involved in thrombolysis. During last two decades, the role of tPA in brain physiology and pathology has been extensively investigated. tPA is expressed in brain regions such as cortex, hippocampus, amygdala and cerebellum, and major neural cell types such as neuron, astrocyte, microglia and endothelial cells express tPA in basal status. After strong neural stimulation such as seizure, tPA behaves as an immediate early gene increasing the expression level within an hour. Neural activity and/or postsynaptic stimulation increased the release of tPA from axonal terminal and presumably from dendritic compartment. Neuronal tPA regulates plastic changes in neuronal function and structure mediating key neurologic processes such as visual cortex plasticity, seizure spreading, cerebellar motor learning, long term potentiation and addictive or withdrawal behavior after morphine discontinuance. In addition to these physiological roles, tPA mediates excitotoxicity leading to the neurodegeneration in several pathological conditions including ischemic stroke. Increasing amount of evidence also suggest the role of tPA in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis even though beneficial effects was also reported in case of Alzheimer's disease based on the observation of tPA-induced degradation of $A{\beta}$ aggregates. Target proteins of tPA action include extracellular matrix protein laminin, proteoglycans and NMDA receptor. In addition, several receptors (or binding partners) for tPA has been reported such as low-density lipoprotein receptor-related protein (LRP) and annexin II, even though intracellular signaling mechanism underlying tPA action is not clear yet. Interestingly, the action of tPA comprises both proteolytic and non-proteolytic mechanism. In case of microglial activation, tPA showed non-proteolytic cytokine-like function. The search for exact target proteins and receptor molecules for tPA along with the identification of the mechanism regulating tPA expression and release in the nervous system will enable us to better understand several key neurological processes like teaming and memory as well as to obtain therapeutic tools against neurodegenerative diseases.

• 생명과학회지
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• 제20권11호
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• pp.1718-1724
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• 2010

누에분말을 과일(키위, 파파야, 파인애플 및 배) 단백질 분해 효소로 반응시켜 생리활성작용 및 이화학적 특성을 조사하기 위하여 pH, 산도, 단백질 함량, 미네랄 함량, 지방산 조성, 단백질 패턴, 항산화 및 혈전용해 활성을 측정하였다. 누에분말은 각 과일 단백질 분해 효소로 $60^{\circ}C$에서 24시간 반응시켰다. 단백질 농도는 과즙액 처리 누에분말에서 약간 높은 것으로 나타났다. 누에분말의 주요 미네랄은 칼륨, 마그네슘, 칼슘 및 아연이였으며, 주요 지방산 조성은 linolenic acid, oleic acid 및 palmitic acid 였다. SDS-PAGE상의 단백질 패턴 분석에서 66-97 kDa 정도 크기의 누에 단백질 밴드가 파인애플, 파파야 및 배 반응에 의해서는 대부분 분해가 일어났지만, 키위 반응에 의해서는 거의 분해가 일어나지 않은 것으로 관찰되었다. 혈전용해 활성은 파파야 및 배 반응에 의한 누에분말에서만 나타났다. 항산화 활성은 0.1% 처리 농도에서 과일 단백질 분해 효소 반응에 의한 누에분말에서 미반응 누에분말 보다 증가하였으나, 시판 항산화제 BHT 처리보다는 활성이 많이 낮았다. 따라서 과즙액 처리 누에분말의 경우 반응 전 누에분말 보다 생리활성작용 및 이화학적 특성이 강화됨으로써 건강기능식품 소재로서의 가치가 증가된 것으로 사료되어 진다.

• BMB Reports
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• 제41권12호
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• pp.858-862
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• 2008

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent proteinase found in cholesterol-rich lipid rafts on the plasma membrane. MT1-MMP hydrolyzes extracellular matrix (ECM) proteins, activates pro-matrix metalloproteinase-2 (proMMP-2) and plays an important role in ECM remodeling, cancer cell migration and metastasis. The role of caveolin-1, an integral protein of caveolae, in the activation of MT1-MMP remains largely unknown. Here, we show that the expression of caveolin-1 attenuates the activation of proMMP-2, reduces proteolytic cleavage of ECM and inhibits cell migration. We utilized the cytoplasmic tail domain deletion (${\Delta}CT$) or the E240A mutant of MT1-MMP. Co-expression of caveolin-1 with the wild-type or the ${\Delta}CT$ MT1-MMP decreased the proMMP-2 activation and inhibited collagen degradation and cell migration. Caveolin-1 had no effect on the catalytically inert E240A MT1-MMP. Our findings suggest that caveolin-1 is essential in the down-regulation of MT1-MMP activity by promoting internalization from the cell surface.