• Journal of Pharmaceutical Investigation
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• 제33권4호
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• pp.273-279
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• 2003

Pancreatin is a enzyme mixture breaking down carbohydrates, proteins and lipids. Most pancreatin used in Korea is imported from foreign countries. However, guideline of each country for pancreatin produced from each country is different. Therefore, guideline for pancreatin imported from several countries, such as Europe, Japan and America, it is standardized to control its quality. Assay of enzyme activity for pancreatin in KP is similar to tat in JP, but it is significantly different from those in FP ad in USP. We measured pancreatin digestive activities of 17 commercial products. Activity assay of digestive enzymes, starch- and lipid-digestive enzymes, for pancreatin by KP method (including JP) was difficult compared to those by FIP ad USP methods. Particularly, activity assays of starch- and lipid-digestive enzymes by KP method were mistakable, ad varied in diluted samples than those by FIP. However, activity assay of protein-digestive enzyme by KP method was similar to that by FIP. Starch-digestive enzyme activities of 17 commercial pancreatins by KP method were lower 0.079-fold compared to those by FIP method. Their protein-digestive enzyme activities by KP method were higher 75.7-fold than those by FIP method. Their lipid-digestive enzyme activities by KP method were lower 0.234-fold compared to those by FIP method.

• 세라미스트
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• 제21권3호
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• pp.204-215
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• 2018

Proteases are one of the most abundant classes of enzymes in living organisms and have been considered major targets for drug development. However, despite the ability to specifically cleave their substrates, many attempts to assay protease activity have generally relied upon the use of gel zymography or fluorophore-labeled peptide substrates, which is limited in rapid and multiplex analysis. Here we review the recent advances in nanoparticle (NP)-utilized assays of protease activity focused on in vitro and cell imaging-based approaches. Owing to large surface area and unprecedented physical properties of NPs, these approaches are anticipated to facilitate many applications related to protease activity-based disease diagnosis and drug discovery.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.

• Applied Biological Chemistry
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• 제42권4호
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• pp.310-316
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• 1999

대두가공제품 중에 존재하는 Bowman-Birk protease inhibitor(BBPI) 함량을 protease 저해활성 측정 및 경합 Enzyme-Linked Immunosorbent Assay(ELISA)로 살펴보았다. 항체제조를 위한 BBPI는 ion exchange chromatography와 전기영동 후 gel slicing 방법으로 시판 soybean trypsin-chymotrypsin inhibitor로부터 순수, 분리하였다. 순수분리한 BBPI를 면역원으로 rabbit anti-BBPI antibody를 조제하였으며 단백질 농도별 titration방법으로 BBP에 비교적 선택적으로 결합하는 항체임을 확인하였다. 이를 이용한 경합 ELISA 방법으로 BBPI를 정량하기 위한 표준 정량곡선을 작성하였으며 시료용액중의 BBPI 함량이 $0.03{\sim}30\;{\mu}g/ml$ 범위일 경우에 정량적인 분석이 가능하였다. 대두품종별 chymotrypsin 저해활성은 $8,462{\sim}12,428\;U/g$이었으며 BBPI 함량은 $482{\sim}692\;mg%$ 이었다. 시판 대두 가공제품 중에서 5종의 콩나물은 건물량 기준으로 $10,695{\sim}13,249\;U/g$의 chymotrypsin 저해활성과 $529{\sim}803\;mg%$의 BBPI 함량을 나타내었으며 일부 두부제품에서도 68.9 mg%정도의 BBPI가 검출되었다. 그 밖의 두유, 된장, 고추장 및 간장 등의 대두발효식품, 탈지대두박 등에서는 chymotrypsin 저해활성 및 BBPI가 거의 검출되지 않았다.

• 대한의생명과학회지
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• 제8권4호
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• pp.245-250
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• 2002

Fibrinolytic enzyme has been purified from the edible mushroom, Tricholoma sejunctum using DEAE-cellulose chromatography, Phenyl-Sepharose chromatography and Mono-S column chromatography. The apparent molecular mass of purified enzyme was estimated to be 17100 Da by SDS-polyacrylamide gel electrophoresis and 19000 Da by gel filtration, Indicating that it was a monomer. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. It has a pH optimum at pH 9.5, suggested that purified enzyme was a alkaline protease. The activity of purified enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that purified enzyme is a metalloprotease. The activity of purified enzyme was increased by Zn$^{2+}$ and Co$^{2+}$, however, the enzyme activity was totally inhibited by Hg$^{2+}$.

• 대한한방내과학회지
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• 제27권4호
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• pp.888-894
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• 2006

Objectives : For the purpose of developing new anti-HIV agents from natural sources, the extracts of Ricinus communis were tested for their inhibitory effects on essential enzymes reverse transcriptase (RT), protease and alpha-glucosidase. Inhibition activity of major compounds of Ricinus communis were predicted from quantitative structure activity relationships (QSAR) in silico. Methods and Results : In the anti-HIV-1 RT using enzyme-linked oligonucleotide sorbent assay (ELOSA) method, water and methanol extracts (100ug/ml) of Ricinus communis showed strong activity of 94.2% and 82.7%, respectively. In the HIV-1 protease and alpha-glucosidase inhibition assay, neither water nor methanol extracts of Ricinus communis inhibited the activity of the enzyme to cleave any substrates as oligopeptides and oligosaccharides. Conclusions : We found that for these samples it is possible that the inhibition of the RT in vitro is due to the secondary metabolites of Ricinus communis such as ricinine and quercetin. It would beof great interest to identify the compounds which are responsible for this inhibition, since all therapeutically useful agents up to date are RT inhibitors.

• BMB Reports
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• 제37권5호
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• KSBB Journal
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• 제29권3호
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• pp.145-154
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• 2014

A fresh vegetable and fruit juice has become a new healthy food available for detoxification, dieting and health. This paper presents the useful information about the quality changes of fresh juice according to different juicer. Quality of fresh juice could be evaluated by several factors such as juice yield, enzyme activity, antioxidant activity, polyphenol contents, and anti-inflammatory activity. The juice yields of 12 different vegetables and fruits were compared using 6 different juicers and it was observed that the yield of slow juicer was better than that of conventional blender. Among 12 samples, the juice yield of grape is the best and the pH of the juice was in the acidic range of 3 and 4. Kiwi and grapefruit were the best in terms of protease enzyme activities by Hemoglobin units on the tyrosine basis and Spectrophotometric acid protease unit and papain units on the tyrosine basis of KFDA protocols. The total polyphenol contents were also high in kiwi and grapefruit. The antioxidant activity by diphenyl-1-picrylhydrazyl (DPPH), superoxide dismutase (SOD), and radical scavenging assay were high in the order of kiwi, grapefruit, grape, tomato, and orange. Anti-inflammatory activities were also assay for 12 samples with 6 juicers. It can be concluded that of fresh fruit and vegetable juice provides a source of antioxidant components and enzymes with high activity. And the enzyme activities could be used as one of the quality indicator of fresh juice. Concerning the juicers used in this study, slow juicer could be recommended to prepare the fresh juice in terms of the juice quality.

• International Journal of Industrial Entomology and Biomaterials
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• 제31권1호
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• pp.30-33
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• 2015

Enzymatic modification of proteins is often used to increase the biological activity of materials. Silkworm powder has been investigated as a functional food resource, but no study has been performed on its modification by commercial food enzyme. Therefore, this study aimed to determine the feasibility of such modification of silkworm powder by alkaline protease. The activity of the enzyme was confirmed using an azocasein assay. Subsequently the silkworm powder was hydrolyzed by enzymatic treatment. UV visible spectrometry showed that the supernatant of silkworm powder subjected to enzymatic treatment had a stronger absorption band than the untreated powder. SDS-PAGE electrophoresis showed that the molecular weight of silkworm powder decreased on enzymatic treatment. Thus the results indicate that commercial enzymes might be used to modify the characteristics of silkworm powder.

• 한국양식학회지
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• 제18권4호
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• pp.245-251
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• 2005

본 연구는 rotifer, B. rotundiformis를 대상으로 소화효소 실험을 하기 위해 이들이 가지고 있는 소화효소의 최고 활성 조건을 확인하기 위해 수행하였다. rotifer, B. rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin 및 TG-lipase는 Tris-HCl buffer 보다 phosphate-NaOH buffer 안정적인 효소활성을 보였다. $\alpha$-amylase, trypsin 및 TG-lipase는 pH 8.0에서, total alkaline proteaset pH 7.0에서 높은 효소 활성을 나타내었다. $\alpha$-amylase 활성은 $40^{\circ}C$에서 가장 높은 활성을 보였으며, total alkaline pretense와 trypsin은 $55{\~}60^{\circ}C$의 온도에서 높은 활성을 나타내었다. 반면 TG-lipase 활성은 $25{\~}30^{\circ}C$의 낮은 온도에서 활성이 높았다. $\alpha$-amylase, total alkaline pretense, trypsin 및 TG-lipase의 활성의 적정 기질 농도는 $3.5\%$ starch, $\0.6%$ azo-casein, $87.5{\mu}M$ BApNA and 81.2 mM olive oil이었다. $\alpha$-amylase, total alkaline protease, trypsin 및 TG-lipase의 활성의 적정 반응시간은 40, 60, 30 and 25 min으로 나타났다. 본 연구 결과에서 얻어진 자료는 rotifer, B. rotundiformis의 소화효소 연구를 위한 기초 자료로 이용될 것이다.