Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs

Abstract

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca²⁺, Co²⁺, Mn²⁺, and DTT. It is inhibited by Fe²⁺, Cd²⁺, Cu²⁺, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Figure 1

Protease assay and phylogenetic tree. (a) Protease activity assay by Skim milk assay agar of Bacillus cereus, (b) The evolutionary history was inferred by using the Maximum Likelihood method using MEGA X software. The tree with the highest log likelihood (− 160,829.40) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches, (c) The PCR amplified gene (1050 bps gene amplified by specific primers), (d) The SDS-PAGE analysis of protein expression. Lane M: Protein ladder. Lane C control: uninduced culture. Lane 1–8: The culture incubation time: 1–8 h. A. The bacterial culture induced with 0.5 mM IPTG incubated at 37, (e) Purified Protein. Lane M-Protein marker, Lane 2-Control, lane3–4 Purified protein in buffer 5 with 200 mM and 500 mM imidazole concentration in elution buffer, respectively, (f) Zymography analysis of purified protein with 1% gelatin as substrate.

Figure 2

(a) Mascot Score Histogram, (b) Phylogenetic tree constructed using amino acid sequence of subtilisin like serine protease by Mega X software, (c) Homologous sequence alignment of protease using Clustal omega program. The catalytic triad is marked with black box.

Figure 3

Structural predictions. (a) I-TASSER structure prediction (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) and image retrieved from Discovery studio software (Discovery Studio Visualizer, v.17.2, San Diego: Dassault Systèmes, 2016), (b) The signal peptide region is colored, Red for N-terminal, Green for H-terminal and Blue for C-terminal (http://www.cbs.dtu.dk/services/SignalIP/), (c) The ligand binding site predicted by I-TASSER (109-HIS,189-TRP, 222-SER, 223-LEU, 224-GLY, 225-SER, 251-ALA, 253-GLY, 254-ASN, 318-THR, 319-SER) (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).

Figure 4

(a) FT-IR spectra of SLSP-k with casein as substrate, (b) Effect of temperature on activity and stability of SLSP-k, (c) Effect of pH on activity.

Figure 5

SEM images. (a) Control chicken feather, (b) Degraded chicken feather by treatment with SLSP-k incubated for 48 h at 50 °C, (c) Control human hair, (d) Degraded human hair by SLSP-k incubated for 72 h at 50 °C.