ArticleMicrobial cell factories2013
Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800.
Article in Microbial cell factories, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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25 citing papers in PubMed, 66 citations in OpenAlex.
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- Characterization of a Nattokinase from the Newly Isolated Bile Salt-ResistantFoods (Basel, Switzerland) · 2022Article
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- Recent Advances in Nattokinase-Enriched Fermented Soybean Foods: A Review.Foods (Basel, Switzerland) · 2022Review
- Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications.Pharmaceutics · 2021Review
- Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.BMC biotechnology · 2021Article
- Homology modeling and heterologous expression of highly alkaline subtilisin-like serine protease from Bacillus halodurans C-125.Biotechnology letters · 2021Article
- Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis.Applied and environmental microbiology · 2020Article
- High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.BMC microbiology · 2019Article
- Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.Probiotics and antimicrobial proteins · 2019Article
- Enhanced NADH Metabolism Involves Colistin-Induced Killing of Bacillus subtilis and Paenibacillus polymyxa.Molecules (Basel, Switzerland) · 2019Article
- Efficient production of extracellular pullulanase in Bacillus subtilis ATCC6051 using the host strain construction and promoter optimization expression system.Microbial cell factories · 2018Article
- Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis.Microbial biotechnology · 2018Article
- Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway inIranian journal of biotechnology · 2018Article
- Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.Applied and environmental microbiology · 2018Article
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3 authors at 1 institution in 1 country.
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Abstract
backgroundNattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production.
resultsA gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of Triton X-100, Tween 20, and Tween 80 showed an activation of Nk up to 141% of its initial activity but SDS strongly inhibited. The enzyme was highly resistant to organic solvents.
conclusionsOur findings demonstrated that an eight-protease-gene-deficient Bacillus subtilis WB800 could overproduce the nattokinase from B. subtilis VTCC-DVN-12-01. Due to high resistance to detergents and organic solvents of this nattokinase, it could be potentially applied in organic synthesis and detergent production.
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