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. 2019 Nov;9(11):391.
doi: 10.1007/s13205-019-1928-9. Epub 2019 Oct 10.

Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties

Abdelnasser S S Ibrahim  1   2 , Yahya B Elbadawi  2 , Mohamed A El-Tayeb  2 , Khalid S Al-Maary  2 , Dina Abdel Fattah Maany  1 , Shebl Salah S Ibrahim  3 , Atif A Elagib  4   5
Affiliations

Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties

Abdelnasser S S Ibrahim et al. 3 Biotech. 2019 Nov.

Abstract

Herein, we report the purification and characterization of an alkaline protease from the alkaliphilic Salipaludibacillus agaradhaerens (formerly Bacillus agaradhaerens) strain AK-R, which was previously isolated from Egyptian soda lakes. The purification procedures resulted in enzyme purification up to 13.3-fold, with a recovery yield of 16.3% and a specific activity of 3488 U/mg protein. AK-R protease was a monomeric protein with an estimated molecular weight of 33.0 kDa. The optimum pH and temperature for AK-R protease were pH 10 and 60 °C, respectively. The enzyme thermostability was significantly enhanced in the presence of CaCl2 by approximately 1.3-fold. Moreover, under optimal conditions, the K m and V max values of the enzyme were 2.63 mg/ml and 4166.7 U/mg, respectively. PMSF caused complete inhibition of the enzyme activity, suggesting that AK-R belongs to the serine protease family. In addition, the enzyme was completely inhibited by EDTA, revealing the requirement of metal ions for AK-R protease activity; hence, it can be classified as a metalloprotease. AK-R protease is a mostly thiol-independent enzyme, since thiol reductants such as β-mercaptoethanol and dithiothreitol had no effect on the enzyme activity. AK-R protease exhibited high stability in several organic solvents, including butanol, amyl alcohol, dimethyl ether, toluene, diethyl ether and methanol. Moreover, AK-R protease showed significant stability to a variety of surfactants and commercial detergents. The features and properties of AK-R alkaline protease are favourable and suggest its potential applications in various industries, particularly in the laundry detergent industry.

Keywords: Alkaline proteases; Alkaliphilic bacteria; Salipaludibacillus agaradhaerens; Soda lakes.

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Conflict of interest statement

Conflict of interestThere is no conflict of interest.

Figures

Fig. 1
Fig. 1
Silver staining of SDS-PAGE gel. M, Protein marker; lane 1: ammonium sulfate fraction; lane 2: purified AK-R protease (Sephadex G-50 fraction)
Fig. 2
Fig. 2
Determination of kinetic parameter values of AK-R alkaline protease. The proteolytic activity was assayed at casein concentrations ranging from 1.0 to 10.0 mg/ml prepared in 50 mM glycine buffer, pH 10, at 60 °C. Km and Vmax values were determined using a Lineweaver–Burk plot
Fig. 3
Fig. 3
Effect of temperature on AK-R alkaline protease activity
Fig. 4
Fig. 4
Effect of pH on the activity of the purified AK-R protease
Fig. 5
Fig. 5
Effect of salinity on the activity of purified AK-R alkaline protease
Fig. 6
Fig. 6
Effect of organic solvents on AK-R protease stability
Fig. 7
Fig. 7
Stability of purified AK-R protease in commercial laundry detergents
See this image and copyright information in PMC

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