본 연구에서는 단백질분해효소활성이 있는 느티만가닥 버섯을 분말 시료화하여 pH, 염도, 온도에 따른 단백질분해효소활성의 변화와 시료 첨가량에 따른 소고기의 육질 변화를 분석하였다. 느티만가닥버섯과 키위의 단백질분해효소 활성을 분석한 결과 느티만가닥버섯은 3.8 unit/ml, 키위가 2.4 unit/ml 로 나타났다. 느티만가닥버섯과 키위 시료를 첨가량별로 소고기에 첨가하였을 때 키위 시료를 첨가한 소고기의 pH는 감소하고 가열감량은 증가한 반면 느티만가닥버섯 시료를 첨가한 소고기의 pH는 첨가량의 증가에 따라 높아졌으며 가열감량은 감소하였다. 절단강도는 첨가량이 증가할수록 감소하였으며 색도에 있어서 L, a, b값 모두 첨가량이 많아질수록 감소하여 느티만가닥버섯의 소 우둔살에 대한 연육효과를 확인하였다. 느티만가닥버섯과 키위 시료를 첨가하여 관능적 품질을 살펴본 결과 느티만가닥버섯 시료의 첨가량이 증가할수록 연육정도에 대한 기호도가 높아졌고 대조구 및 키위시료 첨가구에 비해 전체 적인 기호도가 높았다. 조건에 따른 느티만가닥버섯 시료의 단백질분해효소 활성은 pH는 2 이하, 50 ̊C 이상에서 효소활성이 감소하였고 염농도 조건에서는 1M 이상부터 서서히 떨어지는 경향이었다. 위의 결과로써 질긴 육류를 조리할 때 키위 등 과실 연육재료 대체용으로 느티만가닥버섯이 활용될 가능성이 있음을 보였다.

식물 및 동물성 단백질 유래 펩타이드 형태의 단백질 가수 분해물들은 항산화, 고혈압 완화, 면역조절, 진통완화 및 항균작용 등 생리활성이 있는 것으로 알려져 왔다. 본 연구는 6가지 프로티아제 를 이용하여 오계란 단백질 가수분해물을 생산하고, 생산된 펩타이드의 항산화 능력을 평가하였다. 그 결과 가수분해도의 최대값은 protamex(46.3%)이고, DPPH 라디칼 소거능 최대값은 bromelain(57.23%), 하이드록시 라디칼 소거능 최대값은 alcalase(30.21%), 슈퍼옥사이드 라디칼 소거능 최대값은 alcalase(58.07%), Fe2+ 킬레이션 능력 최대값은 alcalase(72.06%)로 나타났다. 더 나아가 효소별 항산화 저해 능력 IC50 평가하였다. 그 결과 alcalase에 의한 최대값은 금속 킬레이션 눙력(IC50, 1.24 mg/mL) 이고, bromelain에 의한 최대값은 DPPH 소거능(IC50, 2.46 mg/mL)이고, flavourzyme에 의한 최대값은 금속 킬레이션 능력(IC50, 1.25 mg/mL)이고, neutrase에 의한 최대값은 DPPH 소거능(IC50, 3.64 mg/mL)이고, papain에 의한 최대값은 DPPH 소거능(IC50, 3.82 mg/mL)이고, protamex에 의한 최대값 은 DPPH 소거능(IC50, 1.93 mg/mL)이었다. 따라서 protease를 이용하여 오계란 단백질에서 추출한 펩 타이드는 항산화 기능성 식품소재로서 활용할 가치가 높을 것으로 기대한다.

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus. Our previous study indicated that overexpression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. This study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to analyze its inhibitory activity against cysteine protease and physiological role in the parasitism of an endoparsitoid wasp, Cotesia plutellae. The open reading frame (ORF) of CpBV-CST1 encodes a polypeptide of 138 amino acids (15 kDa). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was overexpressed by 0.5 mM IPTG for 4 h. In biological activity assay, partially purified GST-fused rCpBV-CST1 showed inhibitory activity against papain. It also inhibited larval development of P. xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 plays a role in retardation of larval development of P. xylostella during parasitism.

Larvae black soldier fly, Hermetia illucens, is beneficial because its larvae feed on organic materials derived from plants, animals and humans and promote the recycling of food waste and organic materials. Chymotrypsin serine protease is one of the main digestive proteases in the midgut of and is involved in various essential processes. In a previous study, a gene encoding a chymotrypsin-like protease, Hi-SP1, was cloned from the larvae of Hermetia illucens and characterized. The objective of this study was to compare the digestive enzyme activity with various enzymes such as papain, protease and α-chymotrypsin. And also, we investigated the antimicrobial activity of the Hi-SP1 against the spoilage relate bacteria. The growth of the bacteria was inhibited in nutrient broth containing the Hi-SP1.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or typsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, B. thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

This study was carried out to apply an optimized convenient assay, exploiting azo dye-bound chromogenic substrates, to measurement of protease activity. When determined for responses at varying concentrations of two substrates, azocasein and azoalbumin, using 0.5 and 5.0 mg/mL each of bovine pancreas trypsin, 3% azocasein was found to be the most appropriate substrate solution to measure protease activity. Compared with a conventional casein-Folin phenol assay, the chromogen-based protease assay exploiting 3% azocasein showed better precision to have a coefficient of variability in seven repetitive measurements less than 1.11%. When various reagent-grade and industrial proteases that showed proteinase or peptidase activities were tested by this assay at increasing enzyme concentrations, typical shape of rectangular hyperbola in activity-enzyme concentration profiles was observed. In addition, the assay of this study was suitable for activity measurement in real samples that were prepared by hydrolyzing wheat gluten and anchovy fine powder with proteases.

김치 및 젓갈 등의 150여 전통 발효 식품을 시료로 하여 protease 활성을 갖는 유산균을 분리한 결과, 24 U/mgcrude protein의 높은 활성을 갖는 젖산균 BV-26 균주을 분리하였다. API 50CHL kit를 이용하여 BV-26 균주의 당 이용성을 분석하고 16S rRNA 염기서열(99.9% 상동성)을 비교한 결과, 분리된 균주를 L. plantarum BV-26으로 표기하였다. L. plantarum BV-26의 생장과 protease 활성 변화를 MRS 배지를 이용하여 측정한 결과, L. plantarum BV-26의 생장은 배양 6시간 이후 활발하게 진행되어 18시간에 최고의 균체 농도를 보였으며, protease 활성은 배양 후 12시간부터 생성되기 시작하여 16시간에서 최고의 활성을 나타내는 것으로 확인되었다. 따라서 본 연구에서 분리된 L. plantarum BV-26을 동물사료의 발효용 스타터로 이용할 경우 유산균이 갖는 유익한 장점 및 안전성을 확보할 수 있을 뿐만 아니라, 특히 대두박의 발효시 사료의 영양적 가치를 높일 수 있을 것으로 기대된다.

Bee venom contains a variety of protein allergens, including serine proteases. Additionally, bee venom has been used in therapeutic application through immunotherapy for bee venom hypersensitivity and venom therapy as an alternative medicine. Here we present a novel view of the application of bee venom through which bee venom serine protease exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a larger amount of a serine protease as one of its major components. Immunologically, venom serine proteases from bumblebees did not show cross-reactivity with the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. However, Bt-VSP did not activate plasminogen and the fibrinolytic activity of Bt-VSP is less than plasmin. These findings offer insight into the allergic reaction sequence of bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

Background : The purpose of the present investigation is to enhance extracellular acidic protease production by subjecting a protease producing strain Cordyceps pruinosa DK-01 to random mutagenesis by UV irradiation after ethidium bromide treatment. Methods and Results : Mutants were screened as protease producers on the basis of zone of clearance and relative proteolytic activity (RPA) on skimmed milk agar plates. In addition, mutants showed strong pink-red color intensity and different RAPD profiling compared with wild type control. Four mutants were randomly selected and their extracellular enzyme activities were investigated. In liquid culture without casein, 2.2-, 2.9-, 5.2- and 4.4-fold higher acid protease activity was achieved from mutants DK-m9, -m11 and -m12, respectively, than that of wild type strain (11.13 ± 1.60 U/ml). In liquid culture with casein, 1.1-, 1.3-, 1.3 and 1.3-fold higher acid protease activity was achieved with those mutants were found to produce, respectively, than that of wild type strain (93.95 ± 12.84 U/ml). Maximum acid protease activity was noticed from a mutant DK-m11 in liquid culture with casein (121.18 U/ml) and without casein (57.65 U/ml). The extracellular acid protease produced from DJ-m11 that was active in the pH range 4.5-6.5 and optimum temperature for the activity was 37°C. Furthermore, we found a deformed, shorten structure of setae on the elytron surface of dynastid beetles treated with culture supernatant of the DK-m11. Conclusion : These findings have more impact on enzyme economy for biotechnological and insecticidal applications of fungal proteases.

본 연구에서는 새로운 프로바이오틱스를 선발하고자 protease를 생산하는 GRAS균주를 된장 및 청국장으로부터 분리하고 그 특성을 조사하였다. 분리한 균주를 분리 동정 한 결과 각각B. amyloliquefaciens CDD5, B. amyloliquefaciens CPD4 및B. amyloliquefaciens CGD3로 명명하였다. 분리균 주의 생육은 12시간 배양 시, 7.13~7.32 log CFU/mL로 최대 생육도를 보였으며 24시간까지 유지되다가 감소하는 경향 을 나타내었고, 그 중B. amyloliquefaciens CGD3의 protease 활성이9.21 U/mL로서가장높게나타났다. B. amyloliquefaciens 이 생산하는 protease 활성은 pH 7.0~10.0, 온도는 50℃에서 최적활성을 나타내었으며, casein에서 protease활성이 가장 우수하게 나타났다. pH, NaCl, glucose 농도를 달리한 배양 조건에 따른 B. amyloliquefaciens의 생육특성으로는 pH 5.0~10.0의 넓은 범위에서 생육하였고, NaCl은 12% 농도까 지 증식하였으며 glucose 5%까지는 생육하나 10% 농도부 터는 현저하게 생육이 낮아짐을 확인하였다. 또한 혈당저 하를 유도하는 α-glucosidase의 저해활성을 측정한 결과, B. amyloliquefaciens CPD4 및B. amyloliquefaciens CGD3에 서는 각각 96.65% 및 97.85%의 저해율을 나타내었다. 이러 한 결과는 특히 protease활성이 가장 우수하고 α-glucosidase 또한 높은 저해활성을 가진 B. amyloliquefaciens CGD3 균 주의 생육특성을 고려하여 단백질 분해 분야의 프로바이오 틱 적용 및 당뇨예방용 기능성식품 소재로의 가능성이 기대 된다.

우리나라 전통발효식품의 하나인 청국장으로부터 고 비활성 세포외 protease 생산능이 우수한 균주를 분리하고 그 특성을 조사하였다. 분리된 균주 중 D14 균주 배양 상징액의 protease 활성이 15.2 U/mL로서 가장 강하였으며 비활성 또한 40.0 U/mg protein으로서 가장 강하게 나타났다. 이 균주의 특성을 조사한 후 Berge's Manual of Systematic Bacteriology에 준하여 Bacillus subti

Korean medicinal plants were screened for their inhibitory activity against HIV-1 protease. The inhibitory activity of protease was determined by incubating the extracts in reaction mixtures containing protease and substrate His-Lys-Ala-Arg-Val-Leu-(p-NO2-Phe)-Glu-Ala-Nle-Ser-NH2 to perform proteolytic cleavage reactions. In this study the twenty six extracts from medicinal plants were investigated. Of the extracts tested, the extracts from the stem of Morus alba. exhibited the strongest activity with inhibition of 81% at a concentration of 100μg/ml. The extracts of the flower of Saxjfraga stolonifera, and stems of Euonymus japonica and Castanea crenata showed appreciable inhibitory activity (〉50%) against HIV-1 protease at same concentration.

맥류의 한발저항성 반응과 정도 그리고 저항생리적 기작을 구명하기 위하여 발아후 10일된 유묘기(본엽 3매시)에 8일간 단수처리를 하여 질산환원효소와 단백질분해효소의 활성변화 그리고 proline축적을 조사하였던 바 그 결과는 다음과 같다. 1. 전품종의 평균 감소율은 질산환원효소의 활성이 42%이였으며 단백질분해효소의 활성은 73%였으며, 이에 반하여 proline은 대조구에 비하여 단수구가 무려 10배나 증가하였다. 2. 질산환원효소의 활성감소율을 맥종별로 보면 소맥 < 호맥 < 대맥 < 2조대맥ㆍ과맥의 순으로 소맥이 가장 낮았다. 3. 단백질분해효소의 활성감소율을 맥종별로 보면 소맥 < 호맥 < 이조대맥 < 대맥 < 과맥의 순으로 소맥이 높고 과맥이 가장 낮았다. 4. 단수구의 proline의 축적색대량을 맥종별로 보면 소맥 < 대맥 < 호맥 < 과맥 < 이조대맥의 순으로 소맥과 대맥이 높았으며, 대조구에 대한 단수구의 증가비는 호맥(13배) > 소맥ㆍ대맥(11배) > 과맥(9배) > 이조대맥(7배)의 순으로 호맥이 가장 높았다. 5. 한발저항성 과정에서 효소적 및 생리적 대사작용의 관점에서 볼 때 소맥 > 호맥 > 대맥 > 과맥 > 이조대맥의 순으로 한발저항성이 강한 것으로 추정된다.