• Journal of Microbiology and Biotechnology
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• v.10 no.1
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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.

• CELLMED
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• v.4 no.1
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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.

• The Korean Journal of Food And Nutrition
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• v.20 no.4
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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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• v.44 no.10
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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.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.526-531
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• 1988

Procedure for the purification of pretense from culture broth of Streptomyces sp. SMF301 was developed. It was evident that the strain produced two different proteases of which molecular weights were estimated to be 23, 500 and 38, 900 dalton. It was found that the optimum pH of the smaller was 9.0 and that of the larger was 1.0. The optimal temperature of the alkaline pretense was 5$0^{\circ}C$ and that of the neutral pretense was much more stable than neutral protease at extreme condition viz. high temperature, and pH.

• Journal of Microbiology
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• v.34 no.2
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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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• v.3 no.3_4
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.8
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• pp.1353-1358
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• 2004

This study was conducted to increase crude protein and reducing sugar contents in pollen extracts by proteases. Four commercial neutral proteases (Alcalase 2.4L, Protamex, Flavozyme and Protease A) and two alkaline proteases (Protease S and Protease P) were used to prepare acorn and Darae pollen extracts. Contents of moisture, ash, crude protein and crude fat of acorn pollen were 5.2%, 2.7%, 6.2% and 22.3%, respectively, while those of Darae pollen were 5.4%, 2.8%, 1.8% and 27.8%, respectively. Contents of crude protein and reducing sugar in pollen extracts were increased by proteases. Alcalase 2.4L was the most effective in increasing protein contents while Protease A in increasing reducing sugar contents. It is suggested the use of proteases is one of the potential methods for increasing the contents of crude protein and reducing sugar in preparation of pollen extracts.

• BMB Reports
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• v.35 no.2
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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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• v.27 no.3
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• pp.161-170
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• 1989

The proteases of Toxoplasma gcndii were purified partially and characterisrd for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteasfs working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion rxchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotorf. The protease working at pH 6.0 was inactivated by iodoacetamide with LDso of 10-5 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10-5 M and was Promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.