Characterization of a cold-active, detergent-stable metallopeptidase purified from Bacillus sp. S1DI 10 using Response Surface Methodology

Abstract

The colder regions of Earth are inhabited by cold-adapted microorganisms designated as psychrophiles that are known to produce cold-active enzymes, such as peptidases, chaperones, lipases, cellulases, and phosphatases. These types of enzymes are a major part of the market of industrial enzymes. Bacteria isolated from water samples collected from the Chamba region in the Himalayas were screened for peptidase production using skim milk agar plates. Among the peptidase-producing bacteria isolated, 20% of the isolates exhibited fast growth and maximum zones of clearance, and thus, were used for further studies. The 16S rDNA sequence analysis of isolate S1DI 10 identified it as a Bacillus sp. The peptidase was cloned in pET28a vector and expressed in Escherichia coli BL21(DE3) and the His-tagged recombinant protein was purified using Ni-NTA column. The purified peptidase of SIDI 10 was found to be an alkaline, cold-active peptidase with optimal enzyme activity at 10°C and pH 8. An approach of one variable at a time was used to further study the effect of various metal ions, organic solvents and detergents on the peptidase enzyme. The peptidase activity was enhanced in the presence of Fe2+ and Mn2+ (metal ions), hexane (organic solvent), SDS- sodium dodecyl sulfate (anionic detergent) and Tween 80 (nonionic detergent). Response surface methodology (RSM) was used to determine the cumulative effect of these five variables. A 25 full factorial central composite design was applied for the five independent variables to determine the optimal combinations of these constituents at the maximum peptidase activity.

Fig 1. SEM of Bacillus sp. S1DI 10.

Fig 2. Phylogenetic tree of Bacillus sp. S1DI 10 based on 16sRNA gene sequence.

Fig 3. SDS PAGE analysis of purified peptidase expressed in BL21(DE3) strain of E. coli.

(Lane 1—Protein Standard (NEB, USA), Lane 2—Negative Control (E. coli BL21DE3), Lane 3—Uninduced clone, Lane 4—Induced clone, Lane 5—Purified peptidase).

Fig 4. Effect of (A) Temperature (HSD = 4.78, F = 242.04*, p≤0.5) and (B) pH (HSD = 10, F = 701.52*, p≤0.5) on the activity of peptidase isolated from Bacillus sp. S1DI 10.

Fig 5. Effect of various metal ions on the activity of Peptidase produced by Bacillus sp. S1DI10 (F = 8929.03*, p≤0.05).

The residual activity of control was taken as 100%. *Significant at 5% level of significance.

Fig 6. Effect of various organic solvents on the activity of peptidase produced by Bacillus sp. S1DI10 (F = 1394*, p≤0.05).

*Significant at 5% level of significance.

Fig 7. S1DI10 peptidase inhibition by varying concentrations of (A) Detergents (F = 2216.85*, p≤0.05) and (B) Known peptidase inhibitors (F = 676.84*, p≤0.05).

*Significant at 5% level of significance.

Fig 8. Response surface plots of activity of cold peptidase; varying factors were (A) Mn2+ and Fe2+, (B) Mn2+ and Hexane, (C) Mn2+ and SDS, (D) Mn2+ and Tween-80, (E) Fe2+ and Hexane, (F) Fe2+ and SDS, (G) Fe2+ and Tween-80, (H) Hexane and SDS, (I) Hexane and Tween-80, (J) SDS and Tween-80.

Other variables were held at zero level and the residual activity of control was taken as 100%.