Purification and Characterization of a Novel Extracellular Haloprotease Vpr from Bacillus licheniformis Strain KB111

Abstract

Research background: Haloalkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments, are investigated for industrial applications.

Experimental approach: The whole genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS.

Results and conclusions: The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease were 7 and 50 °C, respectively, while the enzyme exhibited maximal activity in the broad salinity range of 2-4 M NaCl. It was fully stable at an alkaline pH range of 7-11 at 50 °C with a half-life of 90 min. Metal ions such as K⁺, Ca²⁺ and Mg²⁺ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acid profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain and a C-terminal domain.

Novelty and scientific contribution: A novel extracellular haloprotease from B. licheniformis was purified, characterized and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.

Keywords: Bacillus sp.; Vpr; halophilic bacteria; serine protease; structural modelling.

Fig. 1

Time course of growth and protease production of Bacillus licheniformis KB111. Enzymatic activity was determined from crude enzyme supernatant using azocasein as a substrate

Fig. 2

Analysis of purified KB-SP by SDS-PAGE (lane 1) and activity staining of gel (lane 2). Lane M=low-molecular-mass marker, lanes 1 and 2=purified KB-SP protein

Fig. 3

Effects of: a) pH, b) temperature, and c) NaCl on protease activity. Enzymatic activity was assayed using Suc-AAPF-pNA as a substrate

Fig. 4

Effects of: a) pH, b) temperature, and c) EDTA on protease stability. Residual activity was determined at 30 °C using Suc-AAPF-pNA as a substrate. For thermal stability against irreversible heat inactivation, residual activity was plotted on a semilog scale against incubation time (b)

Fig. 5

Amino acid sequence alignment of serine proteases. The amino acid sequence of Prepro-KB-SP (KB-SP) is compared with those of Prepro-Bacillus Vpr (BaVpr), Prepro-subtilisin E (BssE) and Prepro-subtilisin Carlsberg (SubC). Dashes represent gaps in the sequence. Black highlights indicate homologous amino acid residues in the three least different proteins. Solid circles indicate a catalytic triad consisting of Asp, His and Ser residues and open circles indicate an oxyanion hole. Signal sequences (underlined) were experimentally determined for BssE (48) and SubC (49) or estimated for KB-SP and BaVpr using the SignalP 5.0 Server (50). Filled arrows represent the position of the propeptides of Pro-BaVpr, Pro-BssE and Pro-SubC, which are autoprocessed. Accession numbers: KUL11341 for KB-SP, AAA22881 for BaVpr, AAA22742 for BssE and X03341 for SubC

Fig. 6

3D model of KB-SP created using Expasy SWISS-MODEL (18) in cartoon representation: a) the main-chain folding of KB-SP and b) the catalytic domain. The catalytic domain (Met133-Glu353 and Val434-Ser569), protease-associated domain (Val354-Leu433) and C-terminal domain (Ser570-Glu778) are coloured green, red and yellow, respectively. The position of the active sites is indicated with a red star and the active sites Asp, His and Ser are shown as purple stick models. N and C=N- and C-terminus, respectively