Bumblebee venom serine protease inhibitors have been shown to inhibit plasmin activity. In this study, a bumblebee (Bombus ignitus) venom serine protease inhibitor (BiVSPI) that acts as an antimicrobial factor was identified. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI was expressed in the venom gland and was present as an 8-kDa peptide in venom. Recombinant BiVSPI expressed in baculovirusinfected insect cells exhibited inhibitory activity against chymotrypsin, but not trypsin. BiVSPI also exhibited inhibitory activity against microbial serine proteases, such as subtilisin A (Ki 6.57 nM) and proteinase K (Ki 7.11 nM), indicating that BiVSPI acts as a microbial serine protease inhibitor. In addition, BiVSPI was also shown to bind directly to Bacillus subtilis, B. thuringiensis, and Beauveria bassiana, but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide novel evidence for the antimicrobial function of this bumblebee venom serine protease inhibitor.

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.

Nuts are one of the most common sources of allergies in individuals of all ages. In order for a particular protein to render an allergic reaction, it must resist proteolytic digestion by intestinal enzymes. In this study, three well-known allergenic nuts, almonds, cashew nuts, and peanuts, were used as samples, and enzyme digestion with Bacillus protease and porcine pepsin was tested. A proteomic approach using two-dimensional gel electrophoresis and an MS/MS analysis was applied to visualize and identify the proteins that were resistant to enzyme digestion. Among the 150 protein spots tested, 42 proteins were assigned functions. Due to the lack of genomic databases, 41% of the identified proteins were grouped as hypothetical. However, 12% of them were well-known allergens, including AraH. The remainder were grouped as storage, enzymes, and binding proteins.

Insect-killing fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana ERL1170 induced melanization of yellow spotted longicorn beetles, Psacothea hilaris as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm, Spodopetra exigua larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment

BCAP 유전자를 지니는 pRB374-BCAP2와 pLip-BCAP2를 B. clausii I-52에 도입 후, 염색체 integration에 의해 구성된 pHPS9-BCAP 형질전환체(B. clausii C5)와 alkaline protease 발현율을 비교하였다. 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO4⋅7H2O 0.01%, FeSO4⋅7H2O 0.05%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침배양(37℃, 48 h, 650 rpm, 1 vvm) 시, pRB374- BCAP2 및 pHPS9-BCAP 형질전환체 각각은 15% 및 61% 정도 alkaline protease 생산이 증가하였다. 그러나 pLip-BCAP2 형질전환체에서는 변화가 없었다. 최고의 활성 균주인, B. clausii C5를 300 L 규모 pilot-scale 액침 배양(37℃, 30 h, 250 rpm, 1 vvm) 시, alkaline protease 생산은 105,685 U/mL로 측정되었다.

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The fungal transformant also shows mammal fibrinolytic activity, by which the transformant can be used pharmaceutically. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

이전 연구에서 protease inhibitor중의 하나인 tannic acid와 파밤나방에 활성이 높은 7 종의 Bacillus thuringiensis에 각각 혼합 처리하여 파밤나방에 살충활성을 실험한 결과, B. thuringiensis subsp. kurstaki KB100에서만 뚜렷한 상승효과를 보였고 tannic acid가 중장액의 protease중 trypsin의 활성을 가장 잘 억제하는 결과를 보였다. 이를 바탕으로 파밤나방 중장액과 trypsin에 대한 tannic acid와 B. thuringiensis strains와의 특성을 비교하고자 파밤나방 중장액 또는 trypsin과 40mM tannic acid를 섞어 반응시킨 후, 7 종의 B. thuringiensis strains parasporal inclusion에 처리하여 단백질의 분해억제 정도를 알아보기 위해 SDS-PAGE를 수행하였다. 그 결과 모든 7 균주의 parasporal inclusion이 약 133kDa크기의 단백질밴드를 나타내는 것으로 보아 분해가 억제된 것을 확인하였다. 반면에 파밤나방 중장액과의 반응에서는 뚜렷한 분해억제 정도를 보인 균은 KB100균주뿐이었다. 그 이외의 6 균주의 parasporal inclusion에서는 약 60~70kDa크기의 단백질밴드를 나타내 중장액에 의해 분해되는 것을 알 수 있다. 따라서 tannic acid는 파밤나방 중장액의 trypsin의 활성을 억제한다고 할 수 있다. 또한. B. thuringiensis KB100균주의 경우에는 다른 6 종의 균주들과는 달리 parasporal inclusion이 tannic acid에 특이적으로 분해가 더욱 억제되어 약 133kDa의 단백질 밴드를 나타낸 것으로 사료된다.

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.

Bee venom is a rich source of pharmacologically active substances. In this study, we characterized a B. terrestris venom Kunitz-type serine protease inhibitor (Bt-KTI). Bt-KTI consists of two exons encoding 82-amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. Bt-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bt-KTI strongly inhibited plasmin, indicating that it acts as a plasmin inhibitor. The electrophoretic mobility shift assay showed that Bt-KTI binds to plasmin, indicating the formation of a plasmin-Bt-KTI complex. These results demonstrate that Bt-KTI acts as an antifibrinolytic agent, suggesting a role for Bt-KTI as an anti-bleeding agent.

Bee venom contains serine proteases and serine protease inhibitors. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen) olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents.

인천 연안 갯벌에서 분리한 호알카리성 Bacillus clausii I-52로부터 세포외 알카리성 단백질 분해효소(BCAP)의 발현 및 생산성을 증가시키기 위하여 BCAP promoter, ribosome 결합 서열, 신호서열, 전구체 서열 및 활성형 BCAP 유전자를 cloning한 재조합 plasmid pHPS9-fuBCAP을 penicliin-protoplast 법으로 B. clausii I-52의 염색체 DNA에 integration 하였고, 도입된 plasmid pHPS9-fuBCAP 유전자는 PCR에 의해 확인하였다. 가장 높은 단백질 분해효소 상대 활성을 보이는 선별된 transformant C5를 생산 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 0.5%, K2HPO4 0.4%, Na2HPO4 0.1%, NaCl 0.4%, MgSO47H2O 0.01%, FeSO47H2O 0.05%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침 배양법(배양온도, 37℃; 배양 시간, 48 h; 교반 속도, 650 rpm; 통기 속도, 1 vvm)으로 배양하여 단백질 분해효소를 발현 및 분비시켰을 때, BCAP 발현 양(134,670 U/ml)은 wild-type(83,960 U/ml)에 비하여 약 1.6 배 증가하였으며, 비활성도(91,611.5 U/mg 단백질)는 wild-type(71,760 U/mg 단백질)에 비하여 약 1.3 배 증가하였다. 또한, B. clausii I-52 염색체 DNA에 integration된 pHPS9-fuBCAP plasmid는 단백질 발현과 함께 8일간의 계대배양 동안에 안정하게 유지되고 있음을 확인하였다.

인천 연안의 심하게 오염된 갯벌로부터 강력한 세포외 알카리성 단백질 분해효소를 생산하는 호알카리 성 Bacillus clausii I-52를 분리하였으며, 이 균주로부터 알카리성 단백질 분해효소의 유전자를 cloning 하여 서열 분석을 하였다. Chromosome 서열이 완전히 밝혀진 Bacillus subtilis의 서열을 기초로 하여 알카리성 단백질 분해효소 및 promoter를 포함하도록 primer를 고안하여 PCR을 수행하여 2,277 bp의 DNA 단편을 얻었으며 BLAST 분석 결과 29 개의 아미노산으로 이루어진 signal peptide, 77 개의 아미 노산으로 이루어진 propeptide 및 275 개의 아미노산을 갖는 활성형의 BCAP으로 구성된 총 381 개의 아미노산을 코딩하는 1,143 bp의 open reading frame을 확인하였다. 활성형 BCAP의 N-말단 아미노산은 Ala이며, 분자량 및 pI 값은 각각 27698.7 Da과 6.30으로 계산되었다. 아미노산 상동성을 분석한 결과, B. subtilis 유래의 nattokinase precursor 및 B. subtilis BSn5 유래의 subtilisin E precursor와 99%의 서열 상동성을 나타내어 B. clausii I-52 유래의 BCAP은 subtilisin 계열의 단백질 분해효소임을 확인하였다. E. coli BL21(DE3)에서 발현한 재조합 BCAP는 N-Suc-Ala-Ala-Pro-Phe-pNA 를 효율적으로 분해하였다. Refolding한 재조합 BCAP은 전형적인 serine protease inhibitor인 PMSF에 의하여 강하게 효소 활성이 억제됨으로써 serine protease 계열의 단백질 분해효소임을 알 수 있었다.

This study was conducted to investigate the effects on in situ ruminal degradation of feed protein sources (soybean meal, P-SBM; dried distillers grain with solubles, P- DDGS; wheat bran, P-WB) treated with protease as compared with conventional feed protein sources (soybean meal, SBM; dried distillers grain with solubles, DDGS; wheat bran, WB). There was no significant change in the chemical composition and amino acid profiles of enzyme treatment protein sources compared with the non-treated groups (p<0.05). But for treatment groups, the solid content and total amino acids were reduced by increasing the moisture content due to proteolytic conditions. On the entire incubation time in situ ruminal degradation rate of dry matter appeared higher in treatment groups compared to control groups (p<0.05), and that of the treatment groups suspended during 48 hours were in the order of P-SBM (97.70%), P-WB (74.26%) and P- DDGS (72.39%). In particular, DM degradation rate of enzyme treated DDGS significantly increased to 43.62%, 45.99%, 55.97%, 69.87% and 72.39%, respectively, incubated during 3, 6, 12, 24 and 48 hours in rumen (p<0.05). Also protein degradation rate of P-WB and P-SBM significantly decreased compared to their respective non-treated sources; however, by contrast, in DDGS it was increased. For P-SBM, protein degradation rate significantly decreased at 6 and 12 hours, and for P-DDGS it was increased at 3 and 6 hours of suspension times in rumen (p<0.05). In particular, protein degradation rate of enzyme treated group suspended for 48 hours were in the order of P-SBM (91.81%), P-WB (86.36%) and P-DDGS (58.87%). This result suggests that protease treatment of feed protein sources might be utilized to increase the bypass ratio into post-rumen for wheat bran, soybean meal, and to improve the utilization of feed protein.

We cloned venom serine proteases from two bumblebee species, Bombus hypocrita sapporoensis and B. ardens ardens. We compared the predicted mature protein sequences of these serine proteasegenes to those previously reported from other bees. Using B. h. sapporoensis venom serine protease(Bs-VSP), we identify that Bs-VSP acts as a fibrin(ogen)olytic enzyme. Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, as demonstrated for B. ignitus and B. terrestrisvenom serine proteases. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic enzyme, providing strong evidence that bumblebee venom serine proteases are hemostatically active proteins that are potentially promising therapeutic agents.

Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tPA. However, it strongly inhibited plasmin, although this inhibitory ability was two-fold weaker than that of aprotinin. The activities of B. ignitusvenom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism for bee venom by which Bi-KTI acts as an antifibrinolytic agent, raising interest in Bi-KTI as a potential clinical agent.

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 protease produced by a Bacillus pumilus CN8 strain was purified by DEAE-Cellulose-52 ion exchange. It has a molecular weight of approximately 96,920 Dalton. In the present study, this protease showed strong activity over a broad range of pH (6.5-9.5) and temperature from 40℃ to 60℃, and the protease performed the maximal activity at pH 7.3 at 42℃. The effect of metal ions on protease activity showed that K+ could slightly increase the protease activity, and other ions such as Zn²+, Fe²+, Na+, Ca²+, Mg²+ had no significant activation or inhibition to the protease (P > 0.05), and the more important is that Cu²+, Mn²+, Sn²+, Cd²+ had a strong inhibitory effect on the protease activity.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Bee venom contains a variety of peptides and enzymes, including serine proteases. Here we describe the molecular cloning and characterization of a serine protease (Bt-VSP) isolated from the venom of the bumblebee Bombus terrestris. The Bt-VSP gene consists of six exons encoding a 358-amino acid protein. The form of Bt-VSP detected in bee venom was the 34-kDa mature protein, which is created by cleavage of the catalytic domain of Bt-proVSP between Arg111 and Val112. 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. The finding that Bt-VSP acts as a fibrin(ogen)olytic enzyme is similar to a previous finding that Bi-VSP, a venom serine protease of B. ignitus, exhibits fibrin(ogen)olytic activity. We also compared major venom components in honeybee and bumblebee, and found that bumblebee venom contains a larger amount of serine protease. Furthermore, unlike bumblebee venom, which exhibits fibrin(ogen)olytic activity owing to the presence of a serine protease, it is likely that honeybee venom lacks fibrin(ogen)olytic activity.