Cloning, Expression, and Structural Elucidation of a Biotechnologically Potential Alkaline Serine Protease From a Newly Isolated Haloalkaliphilic Bacillus lehensis JO-26

Abstract

An alkaline protease gene of Bacillus lehensis JO-26 from saline desert, Little Rann of Kutch, was cloned and expressed in Escherichia coli BL21 (DE3). A 1,014-bp ORF encoded 337 amino acids. The recombinant protease (APrBL) with Asp 97, His 127, and Ser 280 forming catalytic triad belongs to the subtilase S8 protease family. The gene was optimally expressed in soluble fraction with 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG), 2% (w/v) NaCl at 28°C. APrBL, a monomer with a molecular mass of 34.6 kDa was active over pH 8-11 and 30°C-70°C, optimally at pH 10 and 50°C. The enzyme was highly thermostable and retained 73% of the residual activity at 80°C up to 3 h. It was significantly stimulated by sodium dodecyl sulfate (SDS), Ca²⁺, chloroform, toluene, n-butanol, and benzene while completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and Hg²⁺. The serine nature of the protease was confirmed by its strong inhibition by PMSF. The APrBL gene was phylogenetically close to alkaline elastase YaB (P20724) and was distinct from the well-known commercial proteases subtilisin Carlsberg (CAB56500) and subtilisin BPN' (P00782). The structural elucidation revealed 31.75% α-helices, 22.55% β-strands, and 45.70% coils. Although high glycine and fewer proline residues are a characteristic feature of the cold-adapted enzymes, the similar observation in thermally active APrBL suggests that this feature cannot be solely responsible for thermo/cold adaptation. The APrBL protease was highly effective as a detergent additive and in whey protein hydrolysis.

Keywords: Little Rann of Kutch; detergent additive; gene expression; recombinant alkaline protease; structure–function relationship; whey protein hydrolysis.

FIGURE 1

Phylogenetic tree based on a comparison of the APrBL amino acid sequence and some of their closest phylogenetic relatives retrieved from the NCBI database. The tree was reconstructed by the Neighbor-Joining method using MEGA 6 software. The numbers on the tree indicate the percentages of bootstrap support derived from 1,000 replications. The scale bar corresponds to 0.1 substitutions per nucleotide position.

FIGURE 2

(A) Protein expression profile of APrBL. M: 15–150 KD, Lane 1: Preinduced soluble fraction of Escherichia coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 2: Preinduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 3: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 4: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 5: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 6: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 7: Soluble fraction of E. coli BL21 (DE3), Lane 8: Insoluble fraction of E. coli BL21 (DE3), Lane 9: Uninduced soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 10: Uninduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a. (B) Protein purification using Ni-NTA column after expression of the protein in pET28a, 28°C, 0.2 mM IPTG. M: 15–150 KD, L: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, FT: Flow through from the column, E: Column elution in 250 mM imidazole.

FIGURE 3

Effect of NaCl concentration on growth and APrBL activity.

FIGURE 4

Effect of temperature and pH on the activity (A,B) and stability (C,D) of recombinant alkaline protease APrBL, respectively.

FIGURE 5

(A) Secondary structure prediction of APrBL by ENDscript 2.0. On the top are secondary structures (squiggles: helices, arrows: beta strands, TT letters: beta turns). On the bottom, acc denotes bar of accessibility (dark blue: accessible, light blue: intermediate, white: buried) and hyd denotes a bar of hydropathy (pink: hydrophobic, light blue: hydrophilic, white neutral). (B) Three-dimensional (3D) structure showing N-terminal and C-terminal of APrBL generated using I-TASSER server. (C) Ramachandran plot for the predicted 3D structure of APrBL by SWISS-MODEL. A total of 95.22% of the residues are distributed in the most favored region which predicts its stability.

FIGURE 6

Wash performance analysis showing different washing conditions for bloodstain removal.

FIGURE 7

(A) Effect of APrBL enzyme on total protein in milk whey. (B) Effect of APrBL enzyme on degree of hydrolysis of milk whey. (C) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) profile of untreated and enzymatically hydrolyzed milk whey proteins by APrBL enzyme. M: Marker; Lane 1: untreated whey; Lane 2: treated, 1 h; Lane 3: treated, 2 h; Lane 4: treated, 8 h.