Production, Purification, and Biochemical Characterization of a Novel ATP-Dependent Caseinolytic Protease from the Marine Bacterium Cobetia amphilecti KMM 296

蛋白酶 生物化学 PMSF公司 酪蛋白 酶分析 化学 蛋白酵素 基质(水族馆) 生物 生态学
作者
Yulia Noskova,Olga I. Nedashkovskaya,Larissa Balabanova
出处
期刊:Microorganisms [Multidisciplinary Digital Publishing Institute]
卷期号:13 (2): 307-307
标识
DOI:10.3390/microorganisms13020307
摘要

A novel caseinolytic protease (ClpP) of the S14 family from Cobetia amphilecti KMM 296 (CamClpP), comprising 206 amino acids, with a calculated molecular weight of 22.66 kDa and a pI of 4.88, was expressed in Escherichia coli cells to verify the functional annotation of the encoding gene that has low identity with known structures. The proteolytic activity of the purified recombinant enzyme was found to be 2824 U/mg, using 1% casein as a substrate. Enzyme activity was maximal at pH 5.6 and 7.4 in phosphate buffer and was maintained over a wide pH range of 4-10. The optimum temperature for protease activity was 45 °C. The enzyme in its optimal state required the presence of either NaCl or KCl at concentrations of 0.3 and 0.2 M, respectively. The addition of the metal ions Mg2+, Ca2+, Ni2+, Mn2+, Li+, and Zn2+ at 2 mM resulted in a significant inhibition of the protease activity. However, the presence of Co2+ led to a marked activation of the enzyme in the absence of ATP. The enzyme activity was inhibited by ethanol, isopropanol, glycerol, SDS, EGTA, and EDTA. The presence of Triton X-100, acetone, DTT, and PMSF resulted in a significant increase in the CamClpP protease activity. The protease CamClpP effectively and preferentially degrades high-polymer wheat and rye flour proteins. This new proteolytic enzyme with unique properties is of great ecological and biotechnological importance.
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