Difference between revisions of "Part:BBa K1149035"

Latest revision as of 13:36, 11 October 2022

Proteinase K

Encodes a peptidase from proteinase K family, breaks down polylactic acid.

Sequence and Features

Improvement of TJUSLS_China-2022 iGEM team

Proteinase K has two Ca²⁺ bonding sites, which play an important role in maintaining its structural stability. If Ca²⁺ was chelated by EDTA and so on, which is universally used with Proteinase K, the structure of Proteinase K would be unstable and easy to catalyze itself, and it would also influence the thermostability of Proteinase K.
To improve the thermostability of Proteinase K, we employed PyMOL to analyze its 3D structure and designed rationally to mutate its residues to build interactions to replace the function of Ca²⁺. Then we used AlphaFold2 to predict its structure and analyze its stability by FoldX and proved our prediction by experiment. You can see our best thermostability mutation of PK's gene part page in BBa_K4152010, and see our mutation's 3D structure and mutated sites for improvement.
What's more, as a serine protease, Proteinase K has great cytotoxicity, so it has a low production efficiency. So we also improved its expression vector, from E.coli to Pichia Pastoris. To avoid cytotoxicity, we added an α-factor secretion signal to express and secret our Proteinase K extracellularly. And to improve its expression quantity, even more, we used one of the strongest promoters--the AOX1 promoter.
You can watch more information in our composite parts, ranging from BBa_K4152200 to BBa_K4152219.

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 is a member of serine-protease which is synthesized by the fungus Tritirachium album Limber [1] and it consists of 278 amino acids[2]. Proteinase K has the active site of the catalytic triad Asp 39, His 69, Sr224. The optimum pH range for maximal activity of Proteinase K is 7.5–12.0. Its specific activity is about 300 Umg−1. Each unit is capable of hydrolyzing 1 μmole of Ac-Tyr-OEt at 30°C and pH 9.3 per min in the Tris-HCl buffer. Peptidyl chloromethanes and Hg2+ can act as inhibitor for Proteinase K activity[2]

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 [3] 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 [4]

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[5]. 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[5]. 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[5]. 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% [5]

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 [6]. 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[3]. 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 [6]. Inhibition by chloromethyl ketone derivatives, Z-AlaAla-CK and Z-Ala-Phe-CK, just reached a maximum 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 [6].

Reference:

(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

(2) Barret A, Rawlings N, Woessner J. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press. 2012

(3) Fleming D, Rumbaugh K. Approaches to Dispersing Medical Biofilms. Microorganisms. 2017;5(2):15.

(4) 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)

(5) Bajorath J, Hinrichs W, Saenger W. The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem. 1988;176(2):441–7.

(6) 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.