Improved production of alkaline and solvent-stable proteases from a halotolerant Exiguobacterium isolate through heterologous expression

Srivastava, Nitin; Shiburaj, Sugathan; Khare, Sunil Kumar

doi:10.1016/j.ijbiomac.2024.129507

International Journal of Biological Macromolecules

2024

DOI: 10.1016/j.ijbiomac.2024.129507

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Improved production of alkaline and solvent-stable proteases from a halotolerant Exiguobacterium isolate through heterologous expression

Nitin Srivastava,

Sugathan Shiburaj,

Sunil Kumar Khare

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2024

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Cited by 3 publications

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Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4

Majithiya,

Gohel

2024

Appl Biochem Biotechnol

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Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4

Majithiya,

Gohel

2024

Appl Biochem Biotechnol

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Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris

Mushtaq,

Ahmed,

Mehmood

et al. 2024

Biology

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Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6–8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.

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Exiguolysin, a Novel Thermolysin (M4) Peptidase from Exiguobacterium oxidotolerans

Gilmore,

White,

Busetti

et al. 2024

Microorganisms

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This study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of Exiguobacterium oxidotolerans strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C and pH 3, retaining 85% activity at 50 °C, highlighting its potential stability under broad reaction conditions. SDS-PAGE and LC-MS analysis identified the enzyme as a 32 kDa M4-family metalloprotease. Exiguolysin activity was inhibited by 1,10-phenanthroline, confirming its dependence on metal ions for activity. Zymographic analysis and substrate specificity assays revealed selective hydrolysis of matrix metalloproteinase (MMP) substrates but no activity against elastase substrates. Analysis of the predicted gene sequence and structural predictions using AlphaFold identified the presence and position of HEXXH and Glu-Xaa-Xaa-Xaa-Asp motifs, crucial for zinc binding and catalytic activity, characteristic of ‘Glu-zincins’ and members of the M4 peptidase family. High-throughput screening of a 20 × 20 N-alpha mercaptoamide dipeptide inhibitor library against exiguolysin identified SH-CH2-CO-Met-Tyr-NH2 as the most potent inhibitor, with a Ki of 1.95 μM. Notably, exiguolysin selectively inhibited thrombin-induced PAR-1 activation in PC-3 cells, potentially indicating a potential mechanism of virulence in modulating PAR-1 signalling during infection by disarming PARs. This is the first detailed characterization of a peptidase of the M4 (thermolysin) family in the genus Exiguobacterium which may have industrial application potential and relevance as a putative virulence factor.

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Improved production of alkaline and solvent-stable proteases from a halotolerant Exiguobacterium isolate through heterologous expression

Cited by 3 publications

References 36 publications

Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4

Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4

Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris

Exiguolysin, a Novel Thermolysin (M4) Peptidase from Exiguobacterium oxidotolerans

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