Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents

Abstract

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme K_m and V_max values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Figure 1

Phylogenetic tree of rEla from Cohnella sp.A01 and cysteine proteases from different species. The tree was constructed using ClustalW in MEGA X program based on the alignment of the cysteine protease sequences with high similarities.

Figure 2

(a) Protein sequence alignment of rEla and six closely related sequences. Boxed regions show conserved active site residues. (b) Secondary structure of rEla using Phyre2 program. The protease had 39% alpha helix, 28% random coils, 27% beta strand and 6% disordered structures.

Figure 3

Predicted 3D structure of rEla (a) Modeller, (b) SWISS-MODEL and (c) i-Tasser. (d–f) superimpose and optimization of (a, b) structures, (a, c) models and (b, c) models with chimera 1.14. (g) z-score analysis for homology modelled rEla. The plot exhibited the validation of predicted structure. The black dot shows the similarity of model with X-ray and NMR structures. (h) Ramachandran plot revealed that 100% of residues were in favored and allowed regions.

Figure 4

Predicted binding site of cysteine protease in interaction with casein as ligand. Cys 105, His 106, Gly 74 and Trp 75 are predicted catalytic residues.

Figure 5

(a) Docking of rEla and Triton x-100. Triton x-100 is a surfactant with an increasing impact on protease activity. 2D display shows interaction between Ala 32 of rEla with oxygen atom of Triton x-100. (b) Docking of glycerol as a ligand and rEla. Glycerol interacts with allosteric cavity via 5 hydrogen bonds as 2D structure represents. Molecular docking of two cysteine protease specific inhibitors E.64 (c) and Leupeptin (d). E.64 seems to inhibit the enzyme by 6 hydrogen bond interactions with allosteric cavity and changing the protease active conformation. Unlike E.64, Leupeptin is a competitive inhibitor which interacts with active site via 2 hydrogen bonds with His-106 and Glu-14.

Figure 6

MD simulation of rEla cysteine protease in complex with Triton X-100, E.64 and Leupeptin. (a) RMSD graph, (b) RMSF graph and (c) The structure of flexible loop effective on activation/deactivation of rEla. The loop is located between two β-sheets and consists of residues from 39 to 63.

Figure 7

The active site and substrate docking of rEla. The active site residues are shown in purple and catalytic residues are shown in orange. The yellow color shows the substrates. (a) Docked pose for rEla in complexed with Collagen, (b) Active site pocket of rEla with collagen as the substrate, (c) 2D interactions of rEla binding site with collagen. Collagen interacts with rEla through 4 hydrogen bonds with Lys 79, Pro 126 and Arg 155. Hydrogen bonds are shown in green and hydrophobic interactions are pink. (d) Docked pose of rEla in complexed with l.leucine.p.nitroaniline, (e) Active site amino acid residues from docking with l.leucine.p.nitroaniline and (f) 2D interactions of rEla amino acids with l.leucine.p.nitroaniline. The enzyme binds to l.leucine.p.nitroaniline through Cys 105 and Pro 126.

Figure 8

(a) Agarose gel of cloned gen. lane 1: DNA ladder, lane 2: extracted gene from Cohnella sp. A1, lane 3: PCR product, lane 4: pET26-b, lane 5: non-recombinant plasmid, lane 6: recombinant plasmid, lane 7: colony PCR, (b) SDS-PAGE analysis of expressed rEla gene. Lane9: purified rEla. Lane 10: total cellular protein expressed of E. coli BL21.Lane 11: protein molecular mass marker. (c) Casein zymography of rEla (lane 11). (d) rEla resistance against some proteases. Lane 12: proteinase K, lane 13: Trypsin, lane 14–16: rEla, lane 17: protein molecular mass marker. (e) SDS-PAGE analysis of rEla thermal stability after 100 days. Lane 18: enzyme stored at 4 °C, lane 19: − 20 °C with 20% glycerol, lane 20: − 20 °C and lane 21: 25 °C. Lane 22: protein molecular mass marker. Full-length gels are presented in Supplementary Fig. S4 online.

Figure 9

The effect of temperature and pH on rEla. (a) pH profile, the optimum pH for catalytic activity of the enzyme was 8. (b) Temperature profile, the optimum temperature of the enzyme activity was 50 °C. rEla showed more than 70% of residual activity at the temperature range of 20–60 °C (c) pH stability, (d) temperature stability, (e) pH stability at pH 5 and 11 in different times, (f) temperature stability at 50, 70 and 90 °C at different times. (g) Arrhenius plot for E_a^* and (h) E_a^#.

Figure 10

(a) Relative activity of the enzyme during 100 days of storage. rEla showed remarkable stability and the enzyme half-life at room temperature was about 100 days which makes it an excellent candidate for different industrial applications (b) rEla substrates specificity. The enzyme activity in presence of gelatin and collagen was 130% and 120% respectively.

Figure 11

The effect of (a) surfactants, (b) organic solvents, (c) metal ions and (d) inhibitors on rEla relative activity.

Figure 12

The study of enzyme activity in presence of specific inhibitors (a) Leupeptin (b) E.64. Leupeptin is a competitive inhibitor which changes k_m and E.64 is a uncompetitive inhibitor and changes both k_m and v_max of the enzyme.