• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.103-106
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• 2000

Bacillus cereus KCTC 3674 excretes at least two kinds of extracellular proteases into the growth medium. Two major bands of the protease activity with molecular weights of approximately 100 and 38 kDa were obtained after gelatin-SDS-PAGE. The protease with a molecular weight of 38kDa was identified as an extracellular neutral (metallo-) protease. The neutral protease was quite thermostabile but labile to alkaline pH. On the contrary, the 100-kDa protease was thermolabile but stable to alkaline pH. The production of 38-kDa neutral protease was strongly affected by temperature, oxygen, carbonylcyanied m-chlorophenylhydrazone(CCCP) that was defined as a protonophofre, and cerulenin which inhibited lipid synthesis and caused changes in the membrane composition. On the other hand, the production of the 100-kDa protease was strongly affected by only temperature and cerulenin. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing proteases of Bacillus licheniformis, had no effect, whatsoever, on the production of extracellular proteases in B.cereus KCTC 3674.

• 셀메드
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• 제4권1호
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• pp.6.1-6.12
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• 2014

Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

• 한국식품영양학회지
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• 제20권4호
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• pp.378-383
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• 2007

To study the characteristics and processing of Kochujang which is rapidly fermented by commercial enzymes, three kinds of Kochujang(KP-FA, KP-FN, and KP-BN) using commercial proteases and one Kochujang(KM) using Meju were prepared and their qualities investigated. There were only small differences in pH and acidity between each Kochujang. The moisture contents were high tendency in the three kinds of Kochujangs using the commercial proteases at 20 days of fermentation. Reducing sugars had a tendency to decrease during the fermentation in the Kochujangs using the proteases. During the first half of fermentation, the Kochujangs made with proteases showed higher amino nitrogen contents than the Kochujang(KM) made using Meju. Acidic protease activity was high in KP-FA at 20 days of fermentation and neutral protease activity was high in KP-FN and KP-BN at the beginning of fermentation. The Kochujangs made using the proteases, through 20 days of fermentation, obtained high preference in the sensory evaluation for color, texture, and overall acceptability. However, the hot taste was stronger in these Kochujangs during the fermentation.

• BMB Reports
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• 제44권10호
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• pp.665-668
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• 2011

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.526-531
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• 1988

방선균의 단백질 분해효소를 황산 암모늄분획, Sephadex G-75-50 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography, ultrafiltration 등의 과정을 통해 정제하였다. 염기성 단백질 분해 효소의 분자량은 SDS 전기영동에 의해 23,500 dalton 이었으며 Hammarsten casein에 대한 Km값은 0.8g/l였고 이때 Vmax값은 15.1 $\mu$mole/min/mg 이었다. 효소반응 최적 pH는 9.0이었고 최적 반응온도는 5$0^{\circ}C$였다. pH에 대한 안정성은 9.0-10.0 에서 최대로 안정하였고 5$0^{\circ}C$ 이상에서는 효소가 불활성화되었다. 중성단백질 분해효소의 분자량은 38900 dalton 이었으며 Hammarsten casein에 대한 Km값은 0.54g/l였고 이때 Vmax값은 12.4 $\mu$mole/min/mg이었다. 효소반응 최적 pH는 7.0이었고 최적 반응온도는 35$^{\circ}C$였다. pH 7.0-9.0에서는 안정하였으나 4$0^{\circ}C$ 이상에서는 신속하게 불활성화되었다.

• Journal of Microbiology
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• 제34권2호
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• pp.198-205
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• 1996

Two proteases were partially characterized from culture filtrate of Mycobacterium, tuberculosis KIT110. Their molecular weights were approximately 200 and 180 kDa, respectively and they exhibited similar enzymatic characteristics. These enzymes were inhibited significantly by EDTA and to some extent by EGTA. Their activity was enhanced by $Ca^{2+}$ and $Mg^{2+}$ to some degree. However, $Cu^{2+}$ and $Ag^{2+}$ completely inhibited the enzyme activity at the concentration of 2.5 and 5 mM, respectively. The optimal pH was 7.0 and optimal temperature was around $40^{\circ}C$. These enzymes were rapidly inactivated at $80^{\circ}C$. Therefore, they were heat-labile, neutral metalloproteases. These enzymes exhibited antigenicity shown by their reacting with sera from the partients with pulmonary tuberculosis. These enzymes were able to degrade serum proteins including hemoglobin, bovine serum albumin, lysozyme and immunoglobulin G and structural matrix protein such as type I collagen. Therefore, these enzymes may be thought to contribute to tissue necrosis and pathogenesis during infection.

• Fisheries and Aquatic Sciences
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• 제3권3_4호
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• pp.188-194
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• 2000

A protease purified from hepatopancreas of shrimp, Penaeus japonicus, had maximum activity at $70^{\circ}C$ and in neutral and alkaline pH ranges. Specific activity at optimum reaction condition of the protease was estimated to be approximately 12 U/mg/min. The protease was stable in neutral and alkaline pH ranges and activity was retained after heat treatment at $50^{\circ}C$ for 30 min. Apparent $K_m$ and $V_{max}$ value against casein substrate were estimated to be $0.29\%$ and $7.8see^{-1}$, respectively, and those against N-CBZ-L-tyrosine p-nitropheny1 ester (CBZ­Tyr-NE) were 0.38 mM and $2,400 see^{-1}$, respectively. The N-termina1 sequence of the protease showed high homology to the trypsin from same species and the proteases from shrimp. Myosin heavy chain (MHC) from shrimp tail meat was the most susceptible to the protease and actin/tropomyosin were degraded progressively during 4 hr incubation, but to a lesser degree than MHC.

• 한국식품영양과학회지
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• 제33권8호
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• pp.1353-1358
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• 2004

국내산 충매 화분인 도토리 화분과 다래 화분에 단백질 분해효소를 이용하여 화분 추출물을 제조하고, 추출물의 조단백질 함량과 환원당 함량의 변화를 알아보았다. 도토리화분의 일반성분 분석결과 수분 5.2%, 회분 2.7%, 조지방 6.2%, 조단백질 함량은 22.3%였고, 다래화분은 수분 5.4%, 회분 2.8%, 조지방 1.8%, 조단백질 함량은 27.8%였다. Casein을 기질로 사용하여 측정한 효소의 비환성 (specific activity U/mg)은 Protease S>Flavozyme>Alcalase 2.4L>Protamex>Protease P>Protease A의 순이었다. 단백질 분해효소 처리에 의한 화분 추출물의 조단백질 함량은 효소 첨가군이 대조군보다 증가했고, Alcalase 2.4L을 0.2 U, 0.5 U 첨가시에 도토리 화분 추출물은 대조군보다 각각 35.8%, 45.8%, 다래 화분 추출물은 각각 68.8%, 101.5% 증가하였다. 단백질 분해효소 처리에 의한 화분 추출물의 환원당 함량은 효소 첨가군이 대조군보다 증가했고, Protease A를 0.2 U, 0.5 U 첨가 시 도토리 화분 추출물은 대조군보다 각각 11.4%, 18.4%, 다래 화분 추출물은 대조군보다 각각 12.2%, 14.2% 증가하였다. 단백질 분해 효소 처리에 의해 화분 추출물의 조단백질과 환원당의 함량이 증가되었고, 단백질 분해 효소는 화분 추출물의 조단백질 및 환원당 함량을 높이는 방법 중의 하나로 이용될 수 있다고 사료된다.

• BMB Reports
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• 제35권2호
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• pp.143-154
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• 2002

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the $\alpha+\beta$ class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly $\beta$-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

• Parasites, Hosts and Diseases
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• 제27권3호
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• pp.161-170
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• 1989

Toxeplasma의 추출액을 3H-casein을 기질로 반응시켰을 때, pH 6.0과 PH 8.5에서 casein을 분해하였으며, pH 6.0에서는 cysteinyl protease의 억제제 인 iodoacetamide(rAh)에 의해 억제되 었고, 활성제 인 dithiothreitol (DTT)에 의해 환성이 증가하였다. 또 pH 8.5에서는 serine protease의 억제제인 phenylmethylsulfonil fluoride (PMSF)에 의해 활성이 억제되었으며, ATP를 첨가할 때 그 활성이 증가하여 ATP 의존성 효소임을 알 수 있었다. 위의 단백질 분해 효소를 부분 정제하기 위해 여러 chromatography를 실시하였는데, 먼저 DE52 (2.Sfx40 cm)에 통과시켰을 때, 0.05M-0.IM NaCl에 의해 유출되는 분획이 pH 6.0에서 황성을 나타내었으며, 0.25V- 0.3M에서 유출되는 분획이 pH 8.5에서 황성을 나타내었다. 이 분회들을 각각 Sephadex G-200 ($2.50{\phi}{\times}40cm$) 에 통과시켜 pH 6.0에서 활성을 나타내는 분획은 exclusion limit내에서, pH 8.5의 분획은 exclusion limit 외에서 분획을 얻었다. 이들을 각각 hydroxylapatite ($2.50{\phi}{\times}10cm$과 $2.5{\phi}{\times}20cm$)를 통과시켜 각각을 0.05M Phosphate로 유출되는 분회에서 높은 환성을 얻었다. 부분 정제된 분획들의 특성을 검토하기 위하여 억제제를 농도별로 처리하였을 때, pH 0.0에서의 분해 효소는 10-3M IAA에 의해 활성이 반감되어 cysteinyl acid protease임을 알 수 있었다. pH 8.5에서의 분해 효소는 10-5M PMSF에 의해 활성이 반감되었고, ATP에 의해 활성이 증가(ATP의 농도가 2.0mM 이상에서는 억제)하여 ATP-dependent neutral serine protease임을 알 수 있었다.