Microbial proteases are more economical than plant- and animal-derived proteases due to their ease of production and high activity. This study aimed to optimize the production of proteases from fermentative food-derived microorganisms. Five strains with proteolytic activity among 50 Bacillus sp. were first screened. Two strains with high protease activity were identified: Bacillus amyloliquefaciens SRCM 102139 and Bacillus subtilis SRCM 104999. SRCM 102139 strain and SRCM 104999 strain had the highest protease activity in 0.8% glucose and 0.3% yeast extract, and in 0.8% starch and 0.1% soy peptone, respectively. The production of protease for two strains was optimized by the Central Composite Design (CCD) under response surface methodology. The optimal conditions for protease production in SRCM 102139 were 0.5% and 0.347%, pH 6.0, for carbon (glucose) and nitrogen (yeast extract springer 0202) sources, respectively, with a predicted value of 0.929 U/mL. Additionally, the optimal conditions for protease production in SRCM 104999 were 0.5% and 0.5%, pH 6.7, for carbon (starch) and nitrogen (soy peptone HSP-349) sources, respectively, with a predicted value of 0.431 U/mL. The actual protease activities of SRCM 102139 and SRCM 104999 under the established conditions were 0.926 U/mL and 0.428 U/mL, respectively, closely matching the predicted values.

This study was conducted to determine the effects of dietary protein level and supplementation of protease on growth performance, nutrient digestibility, gut microflora, intestinal morphology and fecal noxious gas emission in weanling pigs. A total of 240 weaned pigs (Landrace×Yorkshire×Duroc, 5.82±0.3 kg) were used during 4 weeks in 2 phases (days 0-14, phase 1; and days 15-28, phase 2) feeding program based on age and initial body weight. Pigs were allocated to 2×2 factorial arrangement, including 2 protein levels (HP, high protein; LP, low protein) and 2 protease levels (with or without protease). The average daily gain in the LP treatment (357 g/d) was increased rather than the HP treatment (339 g/d). A greater avarage daily gain was observed in dietary suppiemented protease treatment (358 vs 339 g/d). Average feed intake was greater in the LP treatment (544 g/d) rather than the HP treatment (530 g/d). A greater average daily feed intake was observed in dietary supplemented protease treatment (552 vs 523 g/d). Dry matter and crude protein digestibility were increased in dietary supplemented protease treatment (82.62% and 76.08%, respectively) rather than non-supplemented treatment (81.74% and 75.13%, respectively). Ileal Lactobacillus spp. count increased in dietary supplemented protease treatment (7.42 vs 7.32 log10CFU/g). Emission of H2S was decreased in the LP treatment (4.41 ppm) rather than HP treatment (4.78 ppm). Emission of NH3 was decreased in dietary supplemented protease treatment (10.43 ppm vs 11.76 ppm). In conclusion, the decrease of dietary protein level and supplementation of protease had beneficial effects on growth performance, nutrient digestibility, gut microflora, and noxious gas emission in weanling pigs.

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.

식품첨가물등급의 protease를 이용하여 쌀가루로부터 쌀 전분을 분리하는 효소적 쌀전분 분리·정제법을 구축하기 위해 protease의 반응시간, 반응온도와 농도를 요인으로 하여 변형된 23 완전요인설계법에 따라 protease 반응조건들을 설계하고 이에 따른 쌀전분들의 수율을 조사하였다. 설계된 반응조건들에 따라 제조된 쌀전분들의 수율들에 기초한 반응표면분석을 통해 쌀전분 수율에 대한 protease 반응조건들의 영향을 조사하였다. 또한 효소적 분리·정제법에 의한 쌀전분들의 상업적 활용도를 평가하기 위해 알칼리침지법에 의해 제조된 쌀전분(대조군)과 물리화학적 특성을 대해 비교 분석하였다. Protease를 이용한 효소적 분리· 정제법에 의한 쌀전분들의 수율은 대조군보다 낮았지만 그 상대적 순도는 높은 수준을 나타내었다. Protease에 미량 함유되어 있는 amylase 계통의 효소들에 의한 쌀전분의 부분적인 손상이 예상됨에도 1.5% protease를 이용하여 15℃에서 24시간 동안 처리하여 제조된 쌀전분(RST2)의 아밀로오스 함량은 대조군의 것과 유의적인 차이를 보이지 않았다. 용해도는 효소적 분리·정제법에 의한 쌀전분들이 대조군보다 유의적으로 높은 수준을 나타내었다. 팽윤력은 RST2와 0.5% protease를 이용하여 15℃에서 24시간 동안 처리하여 제조된 쌀전분(RST3)이 대조군과 유의적인 차이를 보이지 않았다. 호화온도는 대조군에 비해 효소적 분리 ·정제법에 의한 쌀전분들이 높은 수준을 나타내었으나 호화엔탈피는 유의적으로 낮은 수준이었다. 페이스팅 점도는 대조군에 비해 효소적 분리·정제법에 의한 쌀전분들이 모든 온도프로파일에 있어 낮은 수준을 나타내었다. 이와 같은 결과들은 효소적 분리·정제법에 사용된 protease에 미량 함유되어 있는 amylase 계통의 효소들에 의한 쌀전분의 부분적인 손상과 protease 처리하는 동안 쌀전분에 있어 annealing이 진행된 결과인 것으로 생각된다. 그럼에도 본 연구에서 효소적 분리·정제법에 의해 제조된 쌀전분들은 높은 고형분 함량을 요구하며, 페이스트의 겔화 또는 노화의 진행이 지연되는 특성을 가공식품의 원료로 적합한 것으로 판단된다. 따라서 효소적 분리·정제법에 의한 쌀전분들은 기존의 알칼리 침지법에 의한 쌀전분과는 다른 특성을 보유한 쌀전분 소재로서 활용가능성이 있을 것으로 생각된다. 또한 효소적 분리·정제법은 알칼리 침지법에 비해 쌀전분의 제조 시간을 단축할 수 있으며, 고농도의 염용액을 배출하지 않아 경제적인 방법인 것으로 판단된다.

Secretory leukocyte protease inhibitor (SLPI), also known as neutrophil elastase and cathepsin-G protease inhibitor, functions in protection of epithelial cells from proteases. SLPI is expressed and secreted by many mucosal tissues, including lungs, seminal vesicles and cervix in women. SLPI plays an important role in protection of endometrial epithelial cells during pregnancy from degradation by degradation by proteases derived from trophoblast at the maternal-conceptus interface. In pigs, SLPI mRNA is known to be expressed in endometrial tissues, but the expression of SLPI in the endometrium throughout the estrous cycle and pregnancy has not been determined. Therefore, we analyzed the expression and regulation of SLPI mRNA in the endometrium throughout the whole stages of the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on Days 0 (day of estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, 30, 60, 90, and 114 of pregnancy. Real-time RT-PCR analysis showed that the expression of SLPI mRNA in the endometrium increases during midt-o late pregnancy. During the estrous cycle, levels of SLPPI mRNA in estrus and proestrus were higher than those in diestrus and metestrus. In situ hybridization analysis showed that SLPI mRNA was specifically localized to the glandular epithelial cells in the endometrium during pregnancy with strong signal intensity during mid-to late pregnancy. SLPI mRNA was not detectable in conceptus tissues on Days 12 and 15 of pregnancy, but SLPI mRNA was expressed in chorioallantoic tissues during mid-to term pregnancy with increasing levels toward term pregnancy. To determine the effects of steroid hormones, estrogen and progesterone, on the expression of SLPI mRNA, endometrial explant tissues from immature pigs were treated with increasing doses of estradiol-17β (E2) and progesterone (P4). Increasing doses of E2 and P4 increased the expression of SLPI mRNA in endometrial tissues. These results showed that SLPI was expressed in the endometrium in a pregnancy stage-and cell type-specific manner and the expression of SLPI was regulated by E2 and P4 in endometrial tissues, suggesting that SLPI may play an important role in regulating the endometrial epithelial cell function during mid-to late pregnancy in pigs. Further analysis to determine the roles of SLPI at the maternal-conceptus interface is still needed.

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated.In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide.Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin,but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however,it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leadingto melanization.The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dualrole of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting andduring the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed inhemocytes and activated in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was activatedin the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reducedBmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticularmelanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, andproPO-activating enzyme. Our findings demonstrate that BmSPH-1 is responsible for the larval-pupal transformation, pupalcuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.

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.

In insects, serine proteases are involved in a variety of physiological processes including digestion, development, and immunity. Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by B. mori lipopolysaccharide-binding protein. Here, we show the dual role of BmSPH-1 in development and immunity of B. mori. BmSPH-1 was expressed in the hemocytes during larval-pupal transformation and localized to the cuticle of silkworms, which indicates that BmSPH-1 is secreted from hemocytes and then transported to the cuticle via the hemolymph. BmSPH-1 was proteolytically activated in the cuticle during larval-pupal transformation and the early pupal stage. BmSPH-1 RNAi resulted in the arrest of larval-pupal transformation and pupal cuticular melanization. Furthermore, the expression of BmSPH-1 was up-regulated in the hemocytes during infection. Taken together, we found that BmSPH-1 is involved in larval-pupal transformation and pupal cuticular melanization as well as the innate immunity of silkworms, which indicates that BmSPH-1 is responsible for either development or immunity.

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the mason bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli, Bacillus subtilis, and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons.

연산오계는 오래전부터 건강기능 증진 및 치료 효능이 높은 것으로 알려져 왔다. 최근 건강 기능식품 소재로 기능성 펩타이드 효능이 알려짐에 따라, 연산오계 다리육으로부 올리고 펩타이드 최적 생산 공정 및 생성물 특성에 대하여 연구를 수행하였다. 최적 효소가수 분해 공정 표면반응 분석을 이 용하여 수행하였다. 최적 공정 조건을 확립하기 위해서 온도 (40, 50, 60℃), pH (pH 6.0, 7.0, 8.0), 효 소 (1, 2, 3%) 범위에서 수행을 하였다. 생성물에 대한 가수분해도, 유리아미노산, 분자량 분포를 분석 하였다. 효소 가수분해 최적 온도는 58℃, pH 7.5, 효소의 농도는 3% 이었다. 최적 조건에서 2 시간 효소 가수분해를 한 결과 75-80% 이었다. 유리 아미노산 총량은 168.131 mg/100 g 이었다. 분자량를 MALDI-TOF 으로 분석을 한 결과 90% 이상이 300-1,000 Da 분포를 보여주었다.

An alkalophilic microorganism, strain DK1122 producing an alkaline protease was identified. DK1122 was isolated from soil collected in central Korea. The strain DK1122 was Gram-positive, 0.7×2-4 μm in size, and its colony was yellowish white, The strain DK1122 was found to be spore-forming, catalase positive, oxidase positive, caseinolytic, and reduce nitrate to nitrite. The protease was produced aerobically on Horikoshi I agar medium (pH 9.0) with 1% (w/v) skim milk at 40°C for 24 h. Through 16S rRNA gene partial sequencing, the strain DK1122 had the 99.7% sequence similarity to 16S rRNA gene sequence of Bacillus pseudofirmus. Based on the biochemical and physiological properties as well as phylogenetic analysis, the isolated strain was named as Bacillus sp. DK1122. It is expected that Bacillus sp. DK1122 may be a promising candidate for a proudcer of an alkaline protease applicable to the food and detergent industries.

1. Pepsin과 pancreatin 소화물들을 비교한 결과, 순수한 팥 단백질 중에서 소화효소 저항성을 가지는 단백질이 존재하는 것으로 확인되었다. 2. 팥의 주요 단백질을 제거하고 pepsin과 pancreatin으로 소화시켰을 때 더 많은 분해가 일어나는 것으로 보아 팥의 주요단백질들 중에 소화효소 저항성을 가지는 것이 많을 것이라 추측된다. 3. 팥의 주요 단백질들은 장 점막세포와는 크게 작용하지 않는 것을 확인할 수 있었다. 4. 팥 단백질의 데이터베이스 구축과 팥 단백질이 다른 영양소들과의 상호작용을 하는 지에 대한 연구가 진행되어야 할 것이다.

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.

Serine protease는 병원체의 표면 melanization, hemolymph coagulation, antimicrobial peptide synthesis 등을 통해 여러 무척추동물의 방어기작을 조절하는것으로 알려 져 있다. 곤충의 경우 Tribolium을 대상으로 이와 같은 연구가 이루어져 왔지만, 혈 액의 량이 그리 많지 않아 연구자들은 최근 갈색거저리(Tenebrio)를 이용하기 시작 하였다. 하지만 아직 유전체(자) 서열정보가 충분하지 않은 상황이다. 본 연구에서 는 이러한 갈색거저리 유충을 이용하여 세포벽이 없으며 사람에서 pneumonia나 다른 호흡기 질환을 일으키는 mycoplasma 와 유사한 acholeplasma lysate를 처리 한 후 접종 전과 후의 전사체의 비교를 통하여 무척추 동물에서의 선천성 면역 관련 유전자들을 동정하고자 하였다. Acholeplasma lysate를 처리하기 전과 후의 각 샘 플들로부터 cDNA library를 구축한 후 random sequencing 을 통해 염기서열을 분 석하였고, 얻어진 서열들로부터 NCBI nr 데이터베이스에 Blastx 분석을 하여 획득 한 서열들을 comparative transcriptomic 방법을 이용하여 분석한 결과, 여러 종류 의 Serine protease 관련 유전자들이 동정되었다. Serine protease (XP_970766.1)의 경우에는 acholeplasma를 처리한 샘플에서 2배 정도 발현이 증가하였고, serine protease P66 (EFA09207.1)의 경우 증감의 변화는 보이지 않았다. 또한 serine protease P146 (EFA04636.1), serine protease H1 (EEZ99180.1) 등의 유전자들도 동정되어 연구하고 있다.

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.