|
|
| Line 5: |
Line 5: |
| | Proteinase K is a member of serine-protease which is synthesized by the fungus Tritirachium album Limber [1] | | Proteinase K is a member of serine-protease which is synthesized by the fungus Tritirachium album Limber [1] |
| | Encodes a peptidase from proteinase K family, breaks down polylactic acid | | Encodes a peptidase from proteinase K family, breaks down polylactic acid |
| − |
| |
| | | | |
| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
| | ===Usage and Biology=== | | ===Usage and Biology=== |
| | | | |
| − |
| |
| − | ==Improvement of UI-Indonesia-2021 iGEM team-- ==
| |
| − | In the absence of a lab, Universitas Indonesia 2021 iGEM team offers literature that gives additional information about Proteinase K
| |
| − |
| |
| − | Proteinase K has the activity to degrade biofilm against several microorganisms including S. aureus, Listeria monocytogenes, Staphylococcus lugdunensis, Staphylococcus haemolyticus, Gardnerella vaginalis, Escherichia coli, Haemophilus influenzae, and Bdellovibrio bacteriovorus [2].Previously, high doses of Proteinase K (25–200 µg/mL) successfully inhibited biofilm formation and degraded preformed biofilm of H. pylori G27 after 24 hours, 37°C [3].
| |
| − |
| |
| − | Proteinase K has two sites Ca 2+ binding. The first binding site (Ca1) consists of Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position. The second binding site (Ca2) consists of the carboxylate of Asp260, the peptide C = 0 of Val16 and two water molecules[4]. Ca2+ ion gives protection against autolysis and increases the thermal stability of proteinase K. Its activity reduces to 20% of original activity within 6 h after Ca 2+ is removed by gel filtration treatment with EDTA. The addition of Ca2+ after 6 h, just restores 28% of its original activity. The loss of Ca2+ ions causes a change in the conformational structure[4]. Autolysis of Proteinase K without Ca 2+ ion happens after incubation >48 h. Removal of Ca2+ decreases the temperature stability of Proteinase K half enzymatic activity from 65°C in the normal state to 46°C[4]. The concentration of 8M urea can decrease Proteinase K activity 65%. Furthermore, Proteinase K also lost its activity when given SDS at a concentration above 12.5% [4] .
| |
| − |
| |
| − | Autolysis does not occur within at least 48h when the concentration of Proteinase K is at or higher than 1.0 mg/ml, but when the concentration is at or below 0.01 mg/ml in an aqueous solution, autolysis occurs slowly after 2h. This condition happens at pH 8 [5]. The optimum temperature of Proteinase K for maximum activity at 37°C and its activity remains constant until 55°C. 20% of methanol can reduces 50% activity of Proteinase K at optimum temperature[5]. Proteinase K shows 90% of original activity when given 10% methanol. But Proteinase K activity can be reduced to only 10% of original activity when 40% methanol is present [5]. Inhibition by chloromethyl ketone derivatives, Z-AlaAla-CK and Z-Ala-Phe-CK, just reached a maximum of 50% of Proteinase K activity at 10 molar excess relative to Proteinase K. Even 25 fold excess synthetic inhibitors cannot increase levels of maximum inhibition activity of Proteinase K. This procedure is carried out in 50 mM Tris-HC1 (pH 8.0) contains 50% methanol [5].
| |
| − |
| |
| − | Reference
| |
| − | <p>(1)EBELING W, HENNRICH N, KLOCKOW M, METZ H, ORTH H, LANG H. Proteinase K from Tritirachium album Limber. European Journal of Biochemistry. 1974;47(1):91-97.</p>
| |
| − | <p>(2) Fleming D, Rumbaugh K. Approaches to Dispersing Medical Biofilms. Microorganisms. 2017;5(2):15.</p>
| |
| − | <p>(3) Windham I, Servetas S, Whitmire J, Pletzer D, Hancock R, Merrell D. Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix. Applied and Environmental Microbiology. 2018;84(14)</p>
| |
| − | <p>(4) Bajorath J, Hinrichs W, Saenger W. The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem. 1988;176(2):441–7.</p>
| |
| − | <p>(5) Bajorath J, Saenger W, Pal GP. Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber. Biochim Biophys Acta BBA - Protein Struct Mol Enzymol. 1988 Jan;954:176–82.</p>
| |
| | | | |
| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
Proteinase K is a member of serine-protease which is synthesized by the fungus Tritirachium album Limber [1]
Encodes a peptidase from proteinase K family, breaks down polylactic acid
